7Beta-hydroxysteroid dehydrogenase mutants and process for the preparation of ursodeoxycholic acid

ABSTRACT

The invention relates to novel 7ß-hydroxysteroid dehydrogenase mutants, to the sequences which encode these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic reactions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCS). The invention also relates to novel processes for the synthesis of UDCS using the enzyme mutants; and to the preparation of UDCS using recombinant, multiply-modified microorganisms.

This application is a continuation of U.S. Ser. No. 13/993,235, which isthe U.S. national phase pursuant to under 35 U.S.C. § 371 of PCTinternational application No. PCT/EP2011/073141, filed Dec. 16, 2011,which claims priority to EP patent application No. 10015726, filed Dec.16, 2012. The entire contents of each of the aforementioned patentapplications are incorporated herein by this reference.

The invention relates to novel 7β-hydroxysteroid dehydrogenase mutants,to the sequences that code for these enzyme mutants, to processes forthe preparation of the enzyme mutants and use thereof in enzymaticreactions of cholic acid compounds, and especially in the preparation ofursodeoxycholic acid (UDCA); the invention also relates to novelprocesses for the synthesis of UDCA using the enzyme mutants; and to thepreparation of UDCA using recombinant, multiply-modified microorganisms.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 11, 2016, isnamed 054410_00062(CON)_SL.txt and is 218,431 bytes in size.

BACKGROUND OF THE INVENTION

The active substances ursodeoxycholic acid (UDCA) and the relateddiastereomer chenodeoxycholic acid (CDCA), among others, have been usedfor many years for the drug treatment of gallstone disease. The twocompounds differ only in the configuration of the hydroxyl group oncarbon atom 7 (UDCA: β-configuration, CDCA: α-configuration). Variousprocesses are described in the prior art for the preparation of UDCA,which are carried out purely chemically or consist of a combination ofchemical and enzymatic process steps. The starting point is in each casecholic acid (CA) or CDCA prepared from cholic acid.

Thus, the classical chemical method for UDCA preparation can berepresented schematically as follows:

A serious disadvantage is, among other things, the following: as thechemical oxidation is not selective, the carboxyl group and the 3α and7α-hydroxyl group must be protected by esterification.

An alternative chemical/enzymatic process based on the use of the enzyme12α-hydroxysteroid dehydrogenase (12α-HSDH) can be represented asfollows and is for example described in PCT/EP2009/002190 of the presentapplicant.

The 12α-HSDH oxidizes CA selectively to 12-keto-CDCA. The two protectionsteps required according to the classical chemical method are thenomitted.

Furthermore, Monti, D., et al., (One-Pot Multienzymatic Synthesis of12-Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule theReaction Equilibria of Five Biocatalysts Working in a Row. AdvancedSynthesis & Catalysis, 2009) describe an alternative enzymatic-chemicalprocess, which can be represented schematically as follows:

The CA is first oxidized from 7α-HSDH from Bacteroides fragilis ATCC25285 (Zhu, D., et al., Enzymatic enantioselective reduction of-ketoesters by a thermostable 7-hydroxysteroid dehydrogenase fromBacteroides fragilis. Tetrahedron, 2006. 62(18): p. 4535-4539) and12α-HSDH to 7,12-diketo-LCA. These two enzymes are each NADH-dependent.After reduction by 7β-HSDH (NADPH-dependent) from Clostridium absonumATCC 27555 (DSM 599) (MacDonald, I. A. and P. D. Roach, Bile inductionof 7 alpha- and 7 beta-hydroxysteroid dehydrogenases in Clostridiumabsonum. Biochim Biophys Acta, 1981. 665(2): p. 262-9), 12-keto-UDCA isformed. The end product is obtained by Wolff-Kishner reduction. Thismethod has the drawback that owing to the position of the equilibrium ofthe catalyzed reaction, a complete reaction is not possible, and thatfor the first step of the reaction it is necessary to use two differentenzymes, which makes the process more expensive. For cofactorregeneration, lactate dehydrogenase (LDH; for regeneration of NAD⁺) andglucose dehydrogenase (GlcDH or GDH, for regeneration of NADPH) areused. A disadvantage with the cofactor regeneration used there is thatthe resultant co-product can only be removed from the reaction mixturewith great difficulty, so that the reaction equilibrium cannot beinfluenced positively, which results in incomplete reaction of theeduct.

A 7β-HSDH from the strain Collinsella aerofaciens ATCC 25986 (DSM 3979;formerly Eubacterium aerofaciens) was described in the year 1982 byHirano and Masuda (Hirano, S. and N. Masuda, Characterization ofNADP-dependent 7 beta-hydroxysteroid dehydrogenases fromPeptostreptococcus productus and Eubacterium aerofaciens. Appl EnvironMicrobiol, 1982. 43(5): p. 1057-63). Sequence information for thisenzyme was not disclosed. The molecular weight determined by gelfiltration was 45 000 Da (cf. Hirano, page 1059, left column).Furthermore, for the enzyme there, the reduction of the 7-oxo group tothe 7β-hydroxyl group was not observed (cf. Hirano, page 1061,Discussion 1st paragraph). A person skilled in the art can therefore seethat the enzyme described by Hirano et al. is not suitable for catalysisof the reduction of dehydrocholic acid (DHCA) in position 7 to3,12-diketo-7β-CA.

The applicant's earlier international patent applicationPCT/EP2010/068576 describes a novel 7β-HSDH from Collinsella aerofaciensATCC 25986, which among other things has a molecular weight (in SDS-gelelectrophoresis) of about 28-32 kDa, a molecular weight (in gelfiltration, in nondenaturing conditions, such as in particular withoutSDS) from about 53 to 60 kDa, and the capacity for stereoselectivereduction of the 7-carbonyl group of 7-keto-LCA to a 7β-hydroxyl group.

In addition, in PCT/EP2010/068576, a process is provided for thepreparation of UDCA, which can be represented schematically as follows:

In this case the oxidation of CA takes place simply, by a classicalchemical route. DHCA is reduced by the pair of enzymes 7β-HSDH and3α-HSDH individually in succession or in one pot to 12-keto-UDCA.Combined with Wolff-Kishner reduction, UDCA can therefore be synthesizedfrom CA in just three steps. 7β-HSDH is dependent on the cofactor NADPH,whereas 3α-HSDH requires the cofactor NADH. The availability of pairs ofenzymes with dependence on the same cofactor or extended dependence(e.g. on the cofactors NADH and NADPH) would be advantageous, becausethis could simplify cofactor regeneration.

The problem to be solved by the invention is to provide further improved7β-HSDHs. In particular, enzyme mutants should be provided, which can beused even more advantageously for enzymatic or microbial preparation ofUDCA via the stereospecific reduction of DHCA in 7-position to3,12-diketo-7β-CA, and in particular have reduced substrate inhibitionand/or have altered cofactor usage (increased, altered specificity orextended dependence).

Another problem is to provide novel enzymatic and microbial synthesisroutes, which in particular are also characterized by simplifiedcofactor regeneration in the reductive preparation of UDCA via DHCA.

SUMMARY OF THE INVENTION

The above problems were solved, surprisingly, by the production andcharacterization of mutants of a novel regio- and stereospecific 7β-HSDHfrom aerobic bacteria of the genus Collinsella, especially of the strainCollinsella aerofaciens and use thereof in the reaction of cholic acidcompounds, especially in the preparation of UDCA.

Furthermore, the above problem was solved by providing a biocatalytic(microbial or enzymatic) process, comprising the enzymatic conversion ofDHCA via two partial reductive steps catalyzed by 7β-HSDH or 3α-HSDH,which can take place simultaneously or with a time delay in any order,to 12-keto-UDCA and cofactor regeneration using dehydrogenases, such asin particular formate dehydrogenase (FDH) enzymes or glucosedehydrogenase (GDH) enzymes, which regenerate the spent cofactor fromthe two partial reductive steps.

DESCRIPTION OF THE FIGURES

FIG. 1A shows the amino acid sequence of 7β-HSDH from Collinsellaaerofaciens and FIG. 1B shows the coding nucleic acid sequence for theamino acid sequence of FIG. 1A; FIG. 1C shows the amino acid sequence of3α-HSDH from Comanomonas testosteroni and FIG. 1D shows the codingnucleic acid sequence for the amino acid sequence of FIG. 1C; FIG. 1Eshows the amino acid sequence of 3α-HSDH from Rattus norvegicus and FIG.1F shows the coding nucleic acid sequence for the amino acid sequence ofFIG. 1E; FIG. 1G shows the coding nucleic acid sequence of the FDHmutant D221G and FIG. 1H shows the amino acid sequence for the nucleicacid sequence of FIG. 1G.

FIG. 2 shows the SDS-gel of a purified 7β-HSDH prepared according to theinvention, with, on lane 1: cell raw extract; lane 2: purified protein;lane M: Page Rouler™, molecular weight marker (Fermentas, Germany).

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D shows the construction schemes of(A) pET21a(+), pET22b(+), pCOLA (Mod) and pET28a(+), respectively.

FIG. 4A and FIG. 4B show the construction schemes of pET21a(+) FDH D221Gand pET21a(+) FDH 7β-HSDH, respectively.

FIG. 5 shows the construction scheme of pCOLA(mod) 3α-HSDH.

FIG. 6A and FIG. 6B show an activity comparison for 7β-HSDH wild typeand the 7β-HSDH mutants G39A and G39S; specifically, Fig. A is a plot ofthe specific enzyme activity versus different substrate concentrationsused at constant cofactor concentration of 100 μM; and Fig. B is a plotof enzyme activity versus different cofactor concentrations used atconstant substrate concentration of 0.3 mM.

FIG. 7 shows the HPLC chromatogram of the biotransformation of DHCS with7β-HSDH and 3α-HSDH in the whole-cell process. It shows the HPLCchromatogram of the whole-cell conversion of the strain E. coli BL21(DE3) hdhA⁻ KanR⁺ pET21a(+) FDH 7β-HSDH pCOLA(mod) 3α-HSDH. The startingconditions selected were 50 mM substrate, 400 mM 15 cosubstrate, pH 6.0.Sampling was carried out after 48 h. The peaks of 12-keto-UDCA (1), anunknown by-product (2), 3,12-diketo-UDCA (3), 7,12-diketo-UDCA (4) andDHCA (5) can be seen.

FIG. 8 shows the construction scheme of the triple vector pET21a(+) FDH7beta (G39A) that bears the coding sequences for FDH D221G, 7β-HSDH G39Aand 3α-HSDH.

FIG. 9 shows the course of whole-cell biotransformation of DHCA. Theproportionate peak areas (according to HPLC analysis) of 12-keto-UDCA,3,12-diketo-UDCA, 7,12-diketo-UDCA and DHCA are shown as a function oftime.

FIG. 10 shows a sequence comparison between the 7β-HSDH wild type andselected mutants. The sequences designated as “7beta-HSDH wild type”,“7beta-HSDH G39D”, “7beta-HSDH G39D R40L”, “7beta-HSDH G39D R40I” and“7beta-HSDH G39D R40V” correspond to SEQ ID NO:2, SEQ ID NO:37, SEQ IDNO:38 and SEQ ID NO:39.

FIG. 11 shows an enzyme-kinetic investigation of 7β-HSDH and the mutantsthereof. The specific enzyme activity is plotted versus differentsubstrate concentrations used (DHCA concentration) at a constantcofactor concentration of 0.1 mM NADPH or 0.5 mM NADH.

FIG. 12 shows a schematic representation of a two-step enzymaticreduction of dehydrocholic acid to 12-keto-ursodeoxycholic acidaccording to the invention, wherein an NADH-dependent 7β-HSDH is usedand a formate dehydrogenase (FDH) is used for cofactor regeneration.

FIG. 13 shows a schematic representation of the principle ofconstruction of the vectors pFr7(D), pFr7(DI), pFr7(DL) and pFr7(DV)that were used for the expression of various NADH-dependent mutants of7β-HSDH.

FIG. 14 shows the proportions of bile salts in biotransformation batchesusing NADH-dependent 7β-HSDH mutants. Results after 24 h process timeand using 100 mM substrate (DHCA) are shown.

FIG. 15 shows a schematic representation of the vector pFr3T7(D).

FIG. 16 shows the result of a whole-cell biotransformation with thestrain E. coli BL49 pFr3T7(D), which comprises the NADH-dependent7β-HSDH (G39D), an NADH-dependent 3α-HSDH and an NADH-dependent FDH. Thebiotransformations were carried out in the following conditions: 20 mLreaction volume, 17.7 g/l_(BTM) cells, 100 mM DHCA, 500 mM ammoniumformate, 26% glycerol, 50 mM MgCl₂, 50 mM KPi buffer (pH 6.5). Duringthe first 5 hours the pH was adjusted manually with formic acid to theinitial value at hourly intervals.

FIG. 17A shows a schematic representation of vector pF(G)r7(A)r3 whichcorresponds to the vector shown in FIG. 8) and FIG. 17B shows aschematic representation of vector pF(G)r7(S)r3.

FIG. 18 shows a schematic representation of the two-step enzymaticreduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid, whereinan NADPH-dependent 7β-HSDH is used and a formate dehydrogenase (FDH)mutant, which regenerates both NADPH− and NADH, is used for cofactorregeneration.

FIG. 19 shows a time-resolved variation of the biotransformation withthe strain E. coli BL49 pF(G)r7(A)r3 at the liter scale. The batchcontained 70 mM DHCA, 17.79 g/l_(BTM) of the stored biocatalyst, 500 mMsodium formate, 26% (v/v) glycerol, 50 mM MgCl₂, in 50 mM KPi buffer (pH6.5). Using pH adjustment, the pH was maintained at pH 6.5 throughoutthe biotransformation.

FIG. 20 shows a schematic representation of the vector p3T7(A)rG.

FIG. 21 shows a schematic representation of the vector p7(A)T3rG.

FIG. 22A and FIG. 22B show a time-resolved variation of thebiotransformation with the strain E. coli BL49 p7(A)T3rG, whichcomprises a GDH for cofactor regeneration. The batch contained 100 mMDHCA, 17.7 g/l_(BTM) of the stored biocatalyst, 500 mM glucose, 10 mMMgCl₂, in 50 mM KPi buffer (pH 7) without (FIG. 22A) and with 0.1 mM NAD(FIG. 22B). The pH was adjusted manually with potassium hydroxidesolution to the initial value.

FIG. 23A shows a schematic representation of vector pF(G)r7(A) and FIG.23B shows a schematic representation of vector pFr3.

FIG. 24 shows a schematic representation of the two-step enzymaticreduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid usingtwo different whole-cell biocatalysts. Reactions A and D are catalyzedby a 7β-HSDH containing whole-cell biocatalyst, and reactions B and Care catalyzed by a 3α-HSDH containing whole-cell biocatalyst. In thecourse of the two-step reaction, the intermediate3,12-diketo-ursodeoxycholic acid must pass from the 7β-HSDH containingwhole-cell biocatalyst into the 3α-HSDH containing whole-cellbiocatalyst, and the intermediate 7,12-diketo-ursodeoxycholic acid mustpass from the 3α-HSDH containing whole-cell biocatalysts into the7β-HSDH containing whole-cell biocatalyst.

FIG. 25 shows the proportions of bile salts of biotransformation batchesafter 24 h when using 70 mM substrate (DHCA). The batches are shown whenusing different proportions of the two biocatalyst strains E. coli BL49pF(G)r7(A) and E. coli BL49 pFr3.

FIG. 26 shows a time-resolved variation of biotransformation with thebiocatalysts E. coli BL49 pF(G)r7(A) and E. coli BL49 pFr3 liter scale.The batch contained 90 mM DHCA, 8.85 g/l_(BTM) E. coli BL49 pF(G)r7(A)and 8.85 g/l_(BTM) E. coli BL49 pFr3, 500 mM ammonium formate, 26% (v/v)glycerol, 50 mM MgCl₂, in 50 mM KPi buffer (pH 6.5). Using pHadjustment, the pH was maintained with formic acid at pH 6.5 throughoutthe biotransformation.

SPECIAL EMBODIMENTS OF THE INVENTION

The invention relates in particular to the following specialembodiments:

1. A 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant, which catalyzesat least the stereospecific enzymatic reduction of a 7-ketosteroid tothe corresponding 7-hydroxysteroid, wherein the mutant has a decreasedsubstrate inhibition (especially for the 7-ketosteroid substrate)compared to the unmutated enzyme, such as in particular an enzymecomprising SEQ ID NO:2 and/or at least the sequence motif VMVGRREaccording to position 36 to 42 thereof; and/or an altered cofactor usage(e.g. increased, altered specificity with respect to a cofactor(especially NADH or NADPH) or an extended dependence, i.e. usage of anadditional cofactor not used previously).2. Mutant according to embodiment 1, which does not display anysubstrate inhibition or which has a K_(i) value for the 7-ketosteroidsubstrate, especially for dehydrocholic acid (DHCA), in the rangefrom >10 mM, e.g. at 11 to 200 mM, 12 to 150 mM, 15 to 100 mM.3. Mutant according to one of the preceding embodiments, wherein thespecific activity (U/mg) in the presence of the cofactor NADPH, comparedto the unmutated enzyme, is raised or lowered by at least 1, 5 or 10%,but especially at least 1-fold, especially 2- to 10-fold; or essentiallyis absent and is replaced by the usage of NADH, or has an extended usageof NADPH and HADH.4. A 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant, which catalyzesat least the stereospecific enzymatic reduction of a 7-ketosteroid tothe corresponding 7-hydroxysteroid, optionally according to one of thepreceding embodiments, wherein the mutant has at least one mutation inthe amino acid sequence according to SEQ ID NO:2 or an amino acidsequence derived therefrom with at least 60% sequence identity, e.g. atleast 65, 70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96,97, 98, 99 or 99.5% to this sequence.5. A 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant, which catalyzesat least the stereospecific enzymatic reduction of a 7-ketosteroid tothe corresponding 7-hydroxysteroid, optionally according to one of thepreceding embodiments, wherein the mutant has at least one mutation inthe sequence motif VMVGRRE according to position 36 to 42 of SEQ ID NO:2or in the corresponding sequence motif of an amino acid sequence derivedtherefrom with at least 60% sequence identity, e.g. at least 65, 70, 75,80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5%to this sequence.6. Mutant according to embodiment 4 or 5, selected froma) the single mutants G39X₁ and R40X₂,b) the double mutants (G39X₁, R40X₂), (R40X₂, R41X₃) and (G39X₁, R41X₃)orc) the triple mutant (G39X₁, R40X₂, R41X₃),(in each case relative to SEQ ID NO:2)wherein X₁, X₂ and X₃ in each case independently of one another standfor any amino acid, especially any, especially natural, amino acid,different from G or R, that decreases substrate inhibition and/ormodifies cofactor usage or cofactor dependence;ord) the corresponding single, double or triple mutants of an amino acidsequence derived from SEQ ID NO:2 with at least 60% sequence identity,e.g. at least 65, 70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94,95, 96, 97, 98, 99 or 99.5% to this sequence.Examples of suitable single mutants comprise: G39A, G39S, G39D, G39V,G39T, G39P, G39N, G39E, G39Q, G39H, G39R, G39K and G39W, and R40D, R40E,R40I, R40V, R40L, R40G, R40A.Examples of suitable double mutants comprise:double combinations of the above G39X₁ and R40X₂ mutants, wherein X₁stands in particular for D or E and/or X₂ can be any amino acid,especially proteinogenic amino acid, such as in particular an amino acidwith aliphatic side chain, for example (G39D,R40I), (G39D,R40L),(G39D,R40V); and the analogous double mutants with G39E instead of G39D;anddouble combinations of the above G39X₁ mutants or R40X₂ mutants withR41X₃ mutants, in which X₃ is any amino acid, especially an, especiallynatural, amino acid, that decreases substrate inhibition and/or thatmodifies cofactor usage or cofactor dependence, different from R, forexample of the type (G39X₁,R41X₃) or (R40X₂,R41X₃),e.g. with X₁=D or E; X₂=I, L or V; X₃=N, I, L or V)for example (R40D,R41I); (R40D,R41L); (R40D,R41V); (R40I,R41I);(R40V,R41I), (R40L,R41I).Examples of suitable triple mutants comprise any triple combinations ofthe above single mutants G39X₁, R40X₂ and R41X₃; wherein X₁, X₂ and X₃are as defined above; but especially wherein X₁ stands for D, or Eand/or X₂ and X₃ stand independently of one another for any amino acid,especially a proteinogenic amino acid, such as in particular triplemutants of the type (G39X₁=D or E; R40X₂=1, L or V; R41X₃=N, I, L or V),for example (G39D,R40I,R41N).Optionally the mutants of embodiments 1 to 6 can have, additionally oralternatively, especially additionally, at least one furthersubstitution, for example 1, 2, 3 or 4 substitutions in the positionsK44, R53, K61 and R64. In this case these residues can be replaced,independently of one another, with any amino acid, especially aproteinogenic amino acid, especially a substitution such that theresultant mutant has a decreased substrate inhibition (especially forthe 7-ketosteroid substrate); and/or has an altered cofactor usage orcofactor dependence (e.g. increased, altered specificity with respect toa cofactor or an extended dependence, i.e. usage of an additionalcofactor not used previously) as defined herein.7. Nucleic acid sequence coding for a 7β-HSDH mutant according to one ofthe preceding embodiments.8. Expression cassette, comprising a nucleic acid sequence according toembodiment 7 under the genetic control of at least one regulatorynucleic acid sequence, and optionally coding sequences for at least one(for example 1, 2 or 3) further enzyme, selected from hydroxysteroiddehydrogenases, especially 3α-HSDH, and dehydrogenases suitable forcofactor regeneration, for example FDH, GDH, ADH, G-6-PDH, PDH. Inparticular the enzymes contained in an expression cassette can usedifferent, but preferably the same pairs of cofactors, for example thepair of cofactors NAD⁺/NADH or NADP⁺/NADPH.9. Vector comprising at least one expression cassette according toembodiment 8.10. Recombinant microorganism, bearing at least one nucleic acidsequence according to embodiment 7 or at least one expression cassetteaccording to embodiment 8 or bearing at least one vector according toembodiment 9 and additionally, optionally, bearing the coding sequencefor a 3α-hydroxysteroid dehydrogenase (3α-HSDH).11. Process for the enzymatic or microbial synthesis of7β-hydroxysteroids, wherein the corresponding 7-ketosteroid is reactedin the presence of a 7β-HSDH mutant according to the definition in oneof the embodiments 1 to 6 or in the presence of a recombinantmicroorganism expressing this mutant according to embodiment 10, and atleast one resultant reduction product is optionally isolated from thereaction mixture.12. The process according to embodiment 11, wherein the ketosteroid tobe reduced is selected from

-   -   a) dehydrocholic acid (DHCA),    -   b) 7-keto-lithocholic acid (7-keto-LCA),    -   c) 7,12-diketo-lithocholic acid (7,12-diketo-LCA) and    -   d) derivatives thereof, such as in particular a salt, amide or        alkyl ester of the acid.        13. The process according to one of the embodiments 11 and 12,        wherein the reduction takes place in the presence of (and with        the consumption of) NADPH and/or NADH.        14. A process for enzymatic or microbial oxidation of        7β-hydroxysteroids, wherein the hydroxysteroid is reacted in the        presence of a 7β-HSDH mutant according to the definition in one        of the embodiments 1 to 6 or in the presence of a microorganism        expressing this mutant according to embodiment 10, and a        resultant oxidation product is optionally isolated from the        reaction mixture.        15. The process according to embodiment 14, wherein the        7β-hydroxysteroid is 3,12-diketo-7β-CA or a derivative thereof,        such as in particular a salt, amide or alkyl ester.        16. The process according to one of the embodiments 14 and 15,        wherein the oxidation takes place in the presence of (and with        the consumption of) NADP⁺ and/or NAD⁺.        17. The process according to one of the embodiments 13 and 16,        wherein the spent redox equivalents are regenerated chemically,        electrochemically or enzymatically, especially in situ.        18. The process according to embodiment 17, wherein spent NADPH        is regenerated by coupling with an NADPH-regenerating enzyme,        wherein this is selected in particular from NADPH        dehydrogenases, alcohol dehydrogenases (ADH), and NADPH        regenerating formate dehydrogenases (FDH), and glucose        dehydrogenase (GDH)-, glucose-6-phosphate dehydrogenase        (G-6-PDH), or phosphite dehydrogenases (PtDH), wherein the        NADPH-regenerating enzyme is optionally expressed by a        recombinant microorganism; or        wherein spent NADH is regenerated by coupling with an        NADH-regenerating enzyme, wherein this is selected in particular        from NADH dehydrogenases, NADH regenerating formate        dehydrogenases (FDH), NADH regenerating alcohol dehydrogenases        (ADH), NADH regenerating glucose-6-phosphate dehydrogenases        (G6PDH), NADH regenerating phosphite dehydrogenases (PtDH) and        NADH regenerating glucose dehydrogenases (GDH), wherein the        NADH-regenerating enzyme is optionally expressed in a        recombinant microorganism.        Expression of the cofactor-regenerating enzyme in a recombinant        microorganism is preferred.        19. The process according to embodiment 18, wherein the        NADPH-regenerating enzyme is selected from natural or        recombinant, isolated or enriched    -   a) alcohol dehydrogenases (EC 1.1.1.2) and    -   b) functional equivalents derived therefrom.        20. The process according to embodiment 18, wherein the        NADPH-regenerating enzyme is selected from mutants of an        NAD⁺-dependent formate dehydrogenase (FDH), which in particular        catalyzes at least the enzymatic oxidation of formic acid to        CO₂, wherein the mutant accepts, compared to the unmutated        enzyme, exclusively or additionally, especially additionally,        NADP⁺ as cofactor; or        wherein the NADH-regenerating enzyme is selected from an        NAD⁺-dependent FDH or an NAD⁺-dependent GDH, such as in        particular an FDH from Mycobacterium vaccae N10, according to        SEQ ID NO:36 and a GDH from Bacillus subtilis according to SEQ        ID NO:48 (which regenerates NADPH and/or NADH) or a modified        form thereof in each case functionally equivalent (with respect        to cofactor regeneration) to the two stated enzymes.        21. The process according to embodiment 20, wherein the FDH        mutant reduces NADP⁺ with a specific activity to NADPH, which        corresponds to about 0.1 to 1000%, such as 1 to 100%, 5 to 80%        or 10 to 50%, of the specific activity of the unmutated        (wild-type) enzyme for the reduction of NAD⁺ to NADH.        22. The process according to one of the embodiments 20 and 21,        wherein the NADP⁺-accepting FDH mutant has at least one mutation        in the amino acid sequence of an FDH from Mycobacterium vaccae        N10 according to SEQ ID NO:36 or an amino acid sequence derived        therefrom with at least 60% sequence identity, e.g. at least 65,        70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97,        98, 99 or 99.5% to this sequence.        23. The process according to one of the embodiments 20 to 22,        wherein the NADP⁺-accepting mutant has at least one mutation in        the sequence motif TDRHRL according to position 221 to 226 of        SEQ ID NO:36 or in the corresponding sequence motif of an amino        acid sequence derived therefrom with at least 60% sequence        identity, e.g. at least 65, 70, 75, 80, 85, or 90, e.g. at least        91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence.        Non-limiting examples of possibly suitable FDH mutants comprise        mutations in the positions D222 and/or R223 of SEQ ID NO:36. As        examples we may mention D222X with X=G, A, K or N; and R223X        with X=H or Y, and combinations of mutations in position 222 and        223.        24. The process according to one of the embodiments 20 to 23,        wherein the NADP⁺-accepting mutant is selected from the single        mutant D222G according to SEQ ID NO:36 (herein also more often        designated as “D221G” mutant (with counting, starting from Ala        in position 2 of SEQ ID NO:36 as first amino acid) or the        corresponding single mutants of an amino acid sequence derived        therefrom with at least 60% sequence identity, e.g. at least 65,        70, 75, 80, 85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97,        98, 99 or 99.5% to this sequence. As concrete examples, we may        mention FDH mutants according to SEQ ID NO: 15, 19 and 35.        Further suitable FDH enzymes are accessible starting from the        wild-type enzymes that can be isolated e.g. from Candida        boidinii or Pseudomonas sp, and insertion of at least one        functional mutation corresponding to the above mutations for        altering the cofactor specificity. Moreover, there have been        numerous studies in the prior art for improving various FDH        properties, such as chemical or thermal stability or catalytic        activity. These are summarized e.g. in Tishkov et al.,        Biomolecular Engineering 23 (2006), 89-110. Thus, single or        multiple point mutations described there can be combined, for        example for increasing enzyme stability, with the mutations        described according to the invention for modified cofactor        usage.        25. A nucleic acid sequence, selected from nucleic acid        sequences a) simultaneously coding for an FDH mutant according        to one of the embodiments 22 to 24 and a 7β-HSDH mutant        according to one of the embodiments 1 to 6 and optionally a        3α-HSDH; or b) simultaneously coding for an FDH mutant according        to one of the embodiments 22 to 24 and an unmutated 7β-HSDH and        optionally a 3α-HSDH; or c) coding for a fusion protein        comprising an FDH mutant according to one of the embodiments 22        to 24 and a 7β-HSDH mutant according to one of the embodiments 1        to 6 and optionally a 3α-HSDH; or d) coding for a fusion protein        comprising an FDH mutant according to one of the embodiments 22        to 24 and an unmutated 7β-HSDH and optionally a 3α-HSDH, e)        simultaneously coding for FDH wild type and a 7β-HSDH mutant        according to one of the embodiments 1 to 6 and optionally a        3α-HSDH; or f) coding for a fusion protein, comprising the FDH        wild type, a 7β-HSDH mutant according to one of the embodiments        1 to 6 and optionally a 3α-HSDH; g) simultaneously coding for a        GDH, a 7β-HSDH wild type and optionally a 3α-HSDH; h) coding for        a fusion protein, comprising a GDH, a 7β-HSDH wild type and        optionally a 3α-HSDH; i) simultaneously coding for a GDH, a        7β-HSDH mutant according to one of the embodiments 1 to 6 and        optionally a 3α-HSDH; and k) coding for a fusion protein,        comprising a GDH, a 7β-HSDH mutant according to one of the        embodiments 1 to 6 and optionally a 3α-HSDH;        wherein the coding sequences can be contained, independently of        one another, singly or multiply in the construct, for example in        2, 3, 4, 5, or 6 to 10 copies. Through selection of the        appropriate copy number, optionally occurring activity        differences in the individual expression products can be        compensated.        26. Expression cassette, comprising a nucleic acid sequence        according to embodiment 25 under the genetic control of at least        one regulatory nucleic acid sequence, wherein the coding        sequences, independently of one another, can be contained singly        or multiply in the construct, for example in 2, 3, 4, 5, or 6 to        10 copies. Through selection of the appropriate copy number,        optionally occurring activity differences in the individual        expression products can be compensated.        27. A vector comprising at least one expression cassette        according to embodiment 26, wherein the coding sequences,        independently of one another, can be contained singly or        multiply in the vector construct, for example in 2, 3, 4, 5, or        6 to 10 copies. Through selection of the appropriate copy        number, optionally occurring activity differences in the        individual expression products can be compensated.        28. A recombinant microorganism bearing at least one nucleic        acid sequence according to embodiment 25 or at least one        expression cassette according to embodiment 27 or bearing at        least one vector according to embodiment 28.        29. A recombinant microorganism that is capable of simultaneous        expression of 7β-HSDH (wild type), an NADP⁺-accepting FDH mutant        and/or the corresponding FDH wild type and optionally of        3α-HSDH; or which is capable of simultaneous expression of        7β-HSDH (wild type), a GDH described herein and optionally a        3α-HSDH described herein.        30. A recombinant microorganism that is capable of simultaneous        expression of a 7β-HSDH mutant, an NADP⁺-accepting FDH mutant        and/or the corresponding FDH wild type and optionally of        3α-HSDH; or which is capable of simultaneous expression of a        7β-HSDH mutant, a GDH described herein and optionally a 3α-HSDH        described herein.        31. The recombinant microorganism according to embodiment 30,        wherein the 7β-HSDH mutant is a mutant according to one of the        embodiments 1 to 6.        32. The recombinant microorganism according to embodiment 29 or        30, wherein the FDH mutant is a mutant according to the        definition in one of the embodiments 20 to 24; and wherein the        FDH wild type is an FDH from Mycobacterium vaccae N10 according        to SEQ ID NO:36 or an FDH derived therefrom with at least 60%        sequence identity, e.g. at least 65, 70, 75, 80, 85, or 90, e.g.        at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% to this        sequence.        33. The recombinant microorganism according to embodiment 29 or        30, wherein the 3α-HSDH is an enzyme comprising an amino acid        sequence according to SEQ ID NO: 6 or 8 or 22 or an amino acid        sequence derived therefrom with at least 60% sequence identity,        e.g. at least 65, 70, 75, 80, 85, or 90, e.g. at least 91, 92,        93, 94, 95, 96, 97, 98, 99 or 99.5% to this sequence.        34. The recombinant microorganism according to one of the        embodiments 29 to 33, bearing the coding sequences for 7β-HSDH,        FDH and 3α-HSDH on one or more (different) expression        constructs. The invention therefore relates to recombinant        microorganisms, which are modified (for example transformed)        with a single-plasmid system, bearing the coding sequences for        7β-HSDH or mutants thereof, FDH or mutants thereof and 3α-HSDH        or mutants thereof in one or more copies, for example in 2, 3,        4, 5, or 6 to 10 copies. The invention therefore also relates to        recombinant microorganisms, which are modified (for example        transformed) with a single-plasmid system, bearing the coding        sequences for 7β-HSDH or mutants thereof, GDH or mutants thereof        and 3α-HSDH or mutants thereof in one or more copies, for        example in 2, 3, 4, 5 or 6 to 10 copies. The enzymes (7β-HSDH,        FDH and 3α-HSDH or mutants thereof) can, however, also be        contained on 2 or 3 separate, mutually compatible plasmids in        one or more copies. Suitable basis vectors for preparing        single-plasmid systems and multicopy plasmids are known by a        person skilled in the art. As examples we may mention, for        single-plasmid system, e.g. pET21a and for multicopy plasmids        e.g. the duet vectors marketed by the company Novagen, such as        pACYCDuet-1, pETDuet-1, pCDFDuet-1, pRSFDuet-1 and pCOLADuet-1.        Vectors of this kind, their compatibility with other vectors and        microbial host strains are given e.g. in the “User Protocol        TB340 Rev. E0305” of the company Novagen.        The optimal combination of enzymes for the generation of plasmid        systems can be undertaken by a person skilled in the art without        undue effort, taking into account the teaching of the present        invention. Thus, a person skilled in the art can, for example,        depending on the cofactor specificity of the 7β-HSDH enzyme used        in each case, select the most suitable enzyme for cofactor        regeneration, selected from the aforementioned dehydrogenases,        especially FDH, GDH and the respective mutants thereof.        Furthermore, there is the possibility of distributing the        enzymes selected for the reaction on two or more plasmids and,        with the resultant plasmids, producing two or more different        recombinant microorganisms, which are then used together for the        biocatalytic reaction according to the invention. The particular        enzyme combination used for preparing the plasmid can in        particular also be applied specifying comparable cofactor usage.        For example, a first microorganism can be modified with a        plasmid, which bears the coding sequence for an NADPH-dependent        7β-HSDH mutant and an FDH mutant regenerating this cofactor        according to the present invention, or which bears the coding        sequence for an NADPH-dependent 7β-HSDH mutant and        NADPH-regenerating GDH, or the coding sequence for an        NADH-dependent 7β-HSDH and an NADH-regenerating FDH and/or GDH.        A second microorganism can, in contrast, be modified with a        plasmid that bears the coding sequence for an NADH-dependent        3α-HSDH and the coding sequence for an NADH-regenerating FDH        wild type and/or for an NADH-regenerating GDH. Both        microorganisms can then be used simultaneously for the        biocatalytic reaction according to the invention.        The use of two separate biocatalysts (recombinant        microorganisms) can offer two essential advantages over the use        of only one biocatalyst, in which all synthesis enzymes are        expressed:        a) The two biocatalysts can be genetically modified and        optimized separately from one another. In particular it is        possible to use different cofactor regeneration enzymes, which        are either optimized for NADH regeneration or for NADPH        regeneration.        b) For the biocatalysis, the biocatalysts can be used in        different proportions. This permits intervention in the        individual reaction rates of the multienzyme process during        biocatalysis, even after all biocatalysts have already been        prepared.        Surprisingly, it was also possible to show, in the context of        the present invention, that the additional membrane transport        steps of the substances that are to react, made necessary by the        use of two biocatalysts, have little or no effect on the        reaction rates, so that these presumed negative aspects are        outweighed by the advantages of the two-cell system.        35. A process for the preparation of ursodeoxycholic acid (UDCA)        of formula (1)

in whichR stands for alkyl, NR¹R², H, an alkali metal ion or N(R³)₄ ⁺, in whichthe residues R³ may be identical or different and stand for H or alkyl,whereina) optionally a cholic acid (CA) of formula (2)

in which R has the meanings given above, is oxidized chemically to thedehydrocholic acid (DHCA) of formula (3)

in which R has the meanings given above;b) DHCA is reduced in the presence of at least one 7β-HSDH mutant(present as isolated enzyme or expressed by a corresponding recombinantmicroorganism) according to the definition in one of the embodiments 1to 6 and in the presence of at least one 3α-hydroxysteroid dehydrogenase(3α-HSDH) (present as isolated enzyme or expressed by a correspondingrecombinant microorganism) to the corresponding 12-keto-ursodeoxycholicacid (12-keto UDCA) of formula (5)

in which R has the meanings given above, (in the presence of and withconsumption of NADH and/or NADPH) and thend) 12-keto-UDCA of formula (5) is reduced chemically to UDCA; ande) the reaction product is optionally further purified.Process step b) can be configured differently. Either both enzymes(7β-HSDH mutant and 3α-HSDH) can be present simultaneously (e.g. one-potreaction with both isolated enzymes or one or more correspondingrecombinant microorganisms are present, which express both enzymes), orthe partial reactions can take place in any order (first the7β-HSDH-mutant-catalyzed reduction and then the 3α-HSDH-catalyzedreduction; or first the 3α-HSDH-catalyzed reduction and then the 7β-HSDHmutant-catalyzed reduction).A process variant for the preparation of UDCA of formula (1) couldtherefore be for example as follows:a) optionally a cholic acid (CA) of formula (2) is oxidized chemically;b) DHCA is reduced in the presence of at least one 7β-HSDH mutant(present as isolated enzyme or expressed by a corresponding recombinantmicroorganism) according to the definition in one of the embodiments 1to 6 to the 3,12-diketo-7β-cholanic acid (3,12-diketo-7β-CA) of formula(4)

(in the presence of and with consumption of NADPH and/or NADH),c) 3,12-diketo-7β-CA is reduced in the presence of at least one3α-hydroxysteroid dehydrogenase (3α-HSDH) (present as isolated enzyme orexpressed by a corresponding recombinant microorganism) to thecorresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)

in which R has the meanings given above, (in the presence of and withconsumption of NADH and/or NADPH, depending on the 3α-HSDH used) andthend) 12-keto-UDCA of formula (5) is reduced chemically to UDCA; ande) the reaction product is optionally further purified.36. The process according to embodiment 35, wherein steps b) and c) arecarried out in the presence of one or more recombinant microorganismsdescribed herein, such as at least one microorganism according toembodiment 10.37. The process according to embodiment 35 or 36, wherein steps b)and/or c) are coupled to identical or different cofactor regenerationsystems (present as isolated enzyme or expressed by a correspondingrecombinant microorganism).38. The process according to embodiment 37, wherein step b) is coupledto a cofactor regeneration system, in which NADPH is regenerated by anNADP⁺-accepting FDH mutant according to the definition in one of theembodiments 20 to 24 with consumption of formic acid or a salt thereof;or is coupled to a cofactor regeneration system, in which NADPH isregenerated by an ADH with consumption of isopropanol; or is coupled toa cofactor regeneration system in which NADPH is regenerated by a GDHwith consumption of glucose; or is coupled to a cofactor regenerationsystem in which spent NADH is regenerated by an NADH regenerating GDH,ADH or FDH.39. The process according to embodiment 37 or 38, wherein step c) iscoupled to a cofactor regeneration step, in which NADPH is regeneratedby an NADP⁺-accepting FDH mutant according to the definition in one ofthe embodiments 20 to 24 with consumption of formic acid or a saltthereof; or wherein step c) is coupled to a cofactor regeneration stepin which NADH is regenerated by an NADH-regenerating FDH mutantaccording to the definition in one of the embodiments 20 to 24 or by theunmutated FDH with consumption of formic acid or a salt thereof or by anNADH-regenerating GDH with consumption of glucose.40. A process for microbial preparation of ursodeoxycholic acid (UDCA)of formula (1)

in whichR stands for alkyl, NR¹R², H, an alkali metal ion or N(R³)₄ ⁺, in whichthe residues R³ may be identical or different and stand for H or alkyl,whereina) optionally a cholic acid (CA) of formula (2)

in which R has the meanings given above, is oxidized chemically to thedehydrocholic acid (DHCA) of formula (3)

in which R has the meanings given above;b) DHCA is reduced in the presence of at least one 7β-HSDH and in thepresence of at least one 3α-HSDH to the corresponding12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)

in which R has the meanings given above, (in the presence of and withconsumption of NADH and/or NADPH) and thenc) 12-keto-UDCA of formula (5) is reduced chemically to UDCA; andd) the reaction product is optionally further purified;wherein the reactions of step b) take place microbially, i.e. in thepresence of whole cells of one or more different recombinantmicroorganisms according to one of the embodiments 28 or 29 to 34,wherein the microorganism or microorganisms carry the enzymes necessaryfor the reaction and cofactor regeneration in a manner described in moredetail herein, or else using at least one nucleic acid sequenceaccording to embodiment 25.For example, DHCA can be reduced in the presence of at least one 7β-HSDHor mutant thereof to 3,12-diketo-7β-cholanic acid (3,12-diketo-7β-CA) offormula (4)

(in the presence of and with consumption of NADPH), and3,12-diketo-7β-CA can be reduced in the presence of at least one3α-hydroxysteroid dehydrogenase (3α-HSDH) or mutant thereof to thecorresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5)

in which R has the meanings given above, (in the presence of and withconsumption of NADH and/or NADPH).Furthermore, however, a reaction sequence is also conceivable,comprising the reduction of DHCA first with 3α-HSDH and the subsequentreduction of the resultant reaction product with 7β-HSDH, as well asboth reaction sequences taking place simultaneously, on the basis of thesimultaneous presence of both HSDHs.41. The process according to embodiment 40, wherein one or more, inparticular one, recombinant microorganism according to one of theembodiments 29 to 34 is used, which for example simultaneously expressesat least one 7β-HSDH mutant according to one of the embodiments 1 to 6,at least one NADP⁺-accepting FDH mutant according to one of theembodiments 20 to 24 and at least one 3α-HSDH, such as in particularaccording to SEQ ID NO: 6, 8 or 22 or mutants thereof in a sequenceidentity of at least about 60%; or which simultaneously expresses atleast one 7β-HSDH mutant according to one of the embodiments 1 to 6, atleast one GDH and at least one 3α-HSDH, such as in particular accordingto SEQ ID NO: 6, 8 or 22 or mutants thereof in a sequence identity of atleast about 60%.42. A bioreactor for carrying out a process according to one of theembodiments 35 to 41, in particular containing at least one of theenzymes (7β-HSDH, FDH, and/or 3α-HSDH or mutants thereof; or 7β-HSDH,GDH and/or 3α-HSDH or mutants thereof) or a recombinant microorganismrecombinantly expressing at least one of these enzymes, especially inimmobilized form.The present invention is not limited to the concrete embodimentsdescribed herein. Rather, a person skilled in the art will be enabled,through the teaching of the present invention, to provide furtherconfigurations of the invention without undue effort. He can, forexample, also purposefully generate further enzyme mutants and screenand optimize these for the desired property profile (improved cofactordependence and/or stability, reduced substrate inhibition); or isolatefurther suitable wild-type enzymes (7β- and 3α-HSDHs, FDHs, GDHs ADHsetc.) and use them according to the invention. Furthermore, for exampledepending on the property profile (especially cofactor dependence) ofthe HSDHs used, such as in particular 7β-HSDH and 3α-HSDH or mutantsthereof, he can select suitable dehydrogenases usable for cofactorregeneration (GDH, FHD, ADH etc.) and mutants thereof, and distributethe selected enzymes to one or more expression constructs or vectors andtherefore if necessary produce one or more recombinant microorganisms,which then make an optimized whole-cell-based method of productionpossible.Further Configurations of the Invention

1. General Definitions and Abbreviations Used

Unless stated otherwise, the term “7β-HSDH” denotes a dehydrogenaseenzyme, which catalyzes at least the stereospecific and/or regiospecificreduction of DHCA or 7,12-diketo-3α-CA (7,12-diketo-LCA) to3,12-diketo-7β-CA or 12-keto-UDCA in particular with stoichiometricconsumption of NADPH, and optionally the corresponding reverse reaction.The enzyme can be a native or recombinantly produced enzyme. The enzymecan basically be mixed with cellular, for example protein impurities,but preferably is in pure form. Suitable methods of detection aredescribed for example in the experimental section given below or areknown from the literature (e.g. Characterization of NADP-dependent 7beta-hydroxysteroid dehydrogenases from Peptostreptococcus productus andEubacterium aerofaciens. S Hirano and N Masuda. Appl Environ Microbiol.1982). Enzymes with this activity are classified under the EC number1.1.1.201.

Unless stated otherwise, the term “3α-HSDH” denotes a dehydrogenaseenzyme that catalyzes at least the stereospecific and/or regiospecificreduction of 3,12-diketo-7β-CA or DHCA to 12-keto-UDCA or7,12-diketo-3α-CA (7,12-diketo-LCA), in particular with stoichiometricconsumption of NADH and/or NADPH, and optionally the correspondingreverse reaction. Suitable methods of detection are described forexample in the experimental section given below or are known from theliterature. Suitable enzymes are obtainable e.g. from Comanomonastestosteroni (e.g. ATCC11996). An NADPH-dependent 3α-HSDH is known forexample from rodents and can also be used. (Cloning and sequencing ofthe cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase,J E Pawlowski, M Huizinga and T M Penning, May 15, 1991, The Journal ofBiological Chemistry, 266, 8820-8825). Enzymes with this activity areclassified under EC number 1.1.1.50.

Unless stated otherwise, the term “GDH” denotes a dehydrogenase enzymethat catalyzes at least the oxidation of β-D-glucose toD-glucono-1,5-lactone with stoichiometric consumption of NAD⁺ and/orNADP⁺ and optionally the corresponding reverse reaction. Suitableenzymes are obtainable e.g. from Bacillus subtilis or Bacillusmegaterium. Enzymes with this activity are classified under EC number1.1.1.47. Unless stated otherwise, the term “FDH” denotes adehydrogenase enzyme that catalyzes at least the oxidation of formicacid (or corresponding formate salts) to carbon dioxide withstoichiometric consumption of NAD⁺ and/or NADP⁺, and optionally thecorresponding reverse reaction. Suitable methods of detection are forexample described in the experimental section given below or are knownfrom the literature. Suitable enzymes are obtainable e.g. from Candidaboidinii, Pseudomonas sp, or Mycobacterium vaccae. Enzymes with thisactivity are classified under EC number 1.2.1.2.

A “pure form” or a “pure” or “substantially pure” enzyme is to beunderstood according to the invention as an enzyme with a degree ofpurity above 80, preferably above 90, especially above 95, and quiteparticularly above 99 wt %, relative to the total protein content,determined by means of usual methods of detecting proteins, for examplethe biuret method or protein detection according to Lowry et al. (cf.description in R. K. Scopes, Protein Purification, Springer Verlag, NewYork, Heidelberg, Berlin (1982)).

A “redox equivalent” means a low-molecular organic compound usable aselectron donor or electron acceptor, for example nicotinamidederivatives such as NAD⁺ and NADH⁺ or their reduced forms NADH and NADPHrespectively. “Redox equivalent” and “cofactor” are used as synonyms inthe context of the present invention. Thus, a “cofactor” in the sense ofthe invention can also be described as “redox-capable cofactor”, i.e. asa cofactor that can be present in a reduced and an oxidized form.

A “spent” cofactor is to be understood as the reduced or oxidized formof the cofactor, which in the course of a specified reduction oroxidation reaction of a substrate is transformed into the correspondingoxidized or reduced form. By regeneration, the oxidized or reducedcofactor form that is formed in the reaction is converted back to thereduced or oxidized starting form, so that it is available again for thereaction of the substrate.

An “altered cofactor usage” is to be understood in the context of thepresent invention as a qualitative or quantitative change compared to areference. In particular, an altered cofactor usage can be observed byundertaking amino acid sequence mutations. This change can then bedetermined compared to the unmutated starting enzyme. Moreover, theactivity with respect to a particular cofactor can be increased orreduced by undertaking a mutation or can be prevented completely. Analtered cofactor usage also comprises, however, changes such thatinstead of a specificity for an individual cofactor, now at least onefurther second cofactor, different from the first cofactor, is usable(i.e. there is an extended cofactor usage). Conversely, however, acapacity for usage of two different cofactors that was originallypresent can be altered so that specificity is only increased for one ofthese cofactors or only reduced for one of these cofactors or iscompletely eliminated. For example, an enzyme that is dependent on thecofactor NAD (NADH) can now, owing to a change of the cofactor usage, bedependent both on NAD (NADH) and the cofactor NADP (NADPH) or theoriginal dependence on NAD (NADH) can be completely transformed to adependence on NADP (NADPH) and vice versa.

The terms “NAD⁺/NADH dependence” or “NADP⁺/NADPH dependence”, unlessstated otherwise, are to be interpreted widely according to theinvention. These terms comprise both “specific” dependences, i.e.exclusively dependence on NAD⁺/NADH or NADP⁺/NADPH, as well as thedependence of the enzymes used according to the invention on bothcofactors, i.e. dependence on NAD⁺/NADH and NADP⁺/NADPH.

This applies correspondingly to the terms “NAD⁺/NADH-accepting” or“NADP⁺/NADPH-accepting”.

The terms “NAD⁺/NADH-regenerating” or “NADP⁺/NADPH-regenerating”, unlessstated otherwise, are to be interpreted widely according to theinvention. These terms comprise both “specific” i.e. exclusive capacityfor regenerating spent cofactor NAD⁺/NADH or NADP⁺/NADPH, and thecapacity for regenerating both cofactors, i.e. NAD⁺/NADH andNADP⁺/NADPH.

“Proteinogenic” amino acids comprise in particular (single-letter code):G, A, V, L, I, F, P, M, W, S, T, C, Y, N, Q, D, E, K, R and H.

“Immobilization” means, according to the invention, the covalent ornoncovalent binding of a biocatalyst used according to the invention,for example a 7β-HSDH on a solid, i.e. essentially insoluble in thesurrounding liquid medium, carrier material. According to the invention,whole cells, such as the recombinant microorganisms used according tothe invention, can correspondingly also be immobilized by means of suchcarriers.

A “substrate inhibition reduced in comparison with the unmutated enzyme”means that the substrate inhibition observed with the unmutated enzymefor a particular substrate is no longer observed, i.e. essentially is nolonger measurable, or only occurs at higher substrate concentration,i.e. the K_(i) value is increased.

“Cholic acid compound” means compounds according to the invention withthe carbon skeleton structure, especially the steroid structure ofcholic acid and the presence of keto and/or hydroxy or acyloxy groups inring position 7 and optionally ring positions 3 and/or 12.

A compound of a special type, for example a “cholic acid compound” or an“ursodeoxycholic acid compound” in particular also means derivatives ofthe underlying starting compound (for example cholic acid orursodeoxycholic acid).

Said derivatives comprise “salts”, for example alkali metal salts suchas lithium, sodium and potassium salts of the compounds; and ammoniumsalts, wherein an ammonium salt comprises the NH₄ ⁺ salt or thoseammonium salts in which at least one hydrogen atom can be replaced witha C₁-C₆-alkyl residue. Typical alkyl residues are, in particular,C₁-C₄-alkyl residues, such as methyl, ethyl, n- or i-propyl-, n-, sec-or tert-butyl, and n-pentyl and n-hexyl and the singly or multiplybranched analogs thereof.

“Alkyl esters” of compounds according to the invention are, inparticular, lower alkyl esters, for example C₁-C₆-alkyl esters. Asnonlimiting examples, we may mention methyl, ethyl, n- or i-propyl, n-,sec- or tert-butyl esters, or longer-chain esters, for example n-pentyland n-hexyl esters and the singly or multiply branched analogs thereof.

“Amides” are, in particular, reaction products of acids according to theinvention with ammonia or primary or secondary monoamines. Such aminesare for example mono- or di-C₁-C₆-alkyl monoamines, wherein the alkylresidues can optionally be further substituted independently of oneanother, for example with carboxyl, hydroxyl, halogen (such as F, C, Br,I), nitro and sulfonate groups.

“Acyl groups” according to the invention are, in particular, nonaromaticgroups with 2 to 4 carbon atoms, for example acetyl, propionyl andbutyryl, and aromatic groups with an optionally substituted mononucleararomatic ring, wherein suitable substituents are selected for examplefrom hydroxyl, halogen (such as F, C, Br, I), nitro and C₁-C₆-alkylgroups, for example benzoyl or toluoyl.

The hydroxysteroid compounds used or prepared according to theinvention, for example cholic acid, ursodeoxycholic acid,12-keto-chenodeoxycholic acid, chenodeoxycholic acid and7-keto-lithocholic acid, can be used in stereoisomerically pure form orin a mixture with other stereoisomers in the process according to theinvention or obtained therefrom. Preferably, however, the compounds usedor prepared are used or isolated in substantially stereoisomericallypure form.

The following table gives the structural formulas, chemical names andthe abbreviations used for important chemical compounds:

Formula Abbreviation Chemical name

CA Cholic acid

DHCA Dehydrocholic acid

3,12-diketo-7β-CA 3,12-Diketo-7β-cholanic acid

12Keto-UDCA 12Keto-ursodeoxycholic acid

UDCA Ursodeoxycholic acid

CA methyl ester Cholic acid methyl ester

3,7-diacetyl-CA- methyl ester 3,7-Diacetyl-cholic acid methyl ester*

12-keto-3,7-diacetyl- CA methyl ester 12-Keto-3,7-diacetyl cholanic acidmethyl ester*

CDCA Chenodeoxycholic acid

7-Keto-LCA 7-Keto-lithocholic acid

7,12-Diketo-LCA 7,12-Diketo-lithocholic acid

12-Keto-CDCA 12-Keto- chenodeoxycholic acid

2. Proteins

The present invention is not limited to the concretely disclosedproteins or enzymes with 7β-HSDH, FDH, GDH or 3α-HSDH activity ormutants thereof, but rather also extends to functional equivalentsthereof.

“Functional equivalents” or analogs of the concretely disclosed enzymesare, in the context of the present invention, polypeptides that aredifferent from them, but still possess the desired biological activity,for example 7β HSDH activity.

For example, “functional equivalents” are to be understood as enzymesthat have, in the test used for 7β-HSDH, FDH, GDH or 3α-HSDH activity,an activity that is higher or lower by at least 1%, e.g. at least 10% or20%, e.g. at least 50% or 75% or 90% than that of a starting enzyme,comprising an amino acid sequence defined herein.

Functional equivalents are in addition preferably stable in the pH rangefrom 4 to 11 and advantageously possess an optimal pH in a pH range from6 to 10, such as in particular 8.5 to 9.5, and an optimal temperature inthe range from 15° C. to 80° C. or 20° C. to 70° C., for example about45 to 60° C. or about 50 to 55° C.

The 7β-HSDH activity can be detected using various known tests. Withoutbeing restricted to this, we may mention a test using a referencesubstrate, e.g. CA or DHCA, under standardized conditions, as defined inthe experimental section.

Tests for determining the FDH, GDH or 3α-HSDH activity are also knownper se.

“Functional equivalents” according to the invention also means, inparticular, “mutants”, which have in at least one sequence position ofthe aforementioned amino acid sequences an amino acid other than thatconcretely stated, but nevertheless possess one of the aforementionedbiological activities. “Functional equivalents” therefore comprise themutants obtainable by one or more, for example 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15, amino acid additions, substitutions,deletions and/or inversions, wherein the stated changes can occur in anysequence position, provided they lead to a mutant with the propertyprofile according to the invention. Functional equivalence in particularalso obtains when the patterns of reactivity between mutant andunaltered polypeptide coincide qualitatively, i.e. for example the samesubstrates are converted at different rates. Examples of suitable aminoacid substitutions are presented in the following table:

Original residue Examples of substitution Ala Ser Arg Lys Asn Gln; HisAsp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val LeuIle; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr ThrSer Trp Tyr Tyr Trp; Phe Val Ile; Leu

“Functional equivalents” in the above sense are also “precursors” of thepolypeptides described and “functional derivatives” and “salts” of thepolypeptides.

“Precursors” are natural or synthetic precursors of the polypeptideswith or without the desired biological activity.

The expression “salts” means both salts of carboxyl groups and acidaddition salts of amino groups of the protein molecules according to theinvention. Salts of carboxyl groups can be prepared in a manner knownper se and comprise inorganic salts, for example sodium, calcium,ammonium, iron and zinc salts, and salts with organic bases, for exampleamines, such as triethanolamine, arginine, lysine, piperidine and thelike. Acid addition salts, for example salts with mineral acids, such ashydrochloric acid or sulfuric acid and salts with organic acids, such asacetic acid and oxalic acid, are also an object of the invention.

“Functional derivatives” of polypeptides according to the invention canalso be prepared on functional amino acid side groups or on their N- orC-terminal end using known techniques. Such derivatives comprise forexample aliphatic esters of carboxylic acid groups, amides of carboxylicacid groups, obtainable by reaction with ammonia or with a primary orsecondary amine; N-acyl derivatives of free amino groups, prepared byreaction with acyl groups; or O-acyl derivatives of free hydroxylgroups, prepared by reaction with acyl groups.

“Functional equivalents” naturally also comprise polypeptides that areobtainable from other organisms, and naturally occurring variants. Forexample, by sequence comparison, homologous sequence regions can befound and equivalent enzymes can be determined based on the concreteinstructions of the invention.

“Functional equivalents” also comprise fragments, preferably individualdomains or sequence motifs, of the polypeptides according to theinvention, which for example have the desired biological function.

“Functional equivalents” are in addition fusion proteins that have oneof the aforementioned polypeptide sequences or functional equivalentsderived therefrom and at least one further, functionally differenttherefrom, heterologous sequence in functional N- or C-terminal linkage(i.e. without mutual substantial functional impairment of the fusionprotein parts). Nonlimiting examples of said heterologous sequences aree.g. signal peptides, histidine anchors or enzymes.

“Functional equivalents” that are also included according to theinvention are homologs of the concretely disclosed proteins. Thesepossess at least 60%, preferably at least 75%, especially at least 85%,for example 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology (oridentity) to one of the concretely disclosed amino acid sequences,calculated according to the algorithm of Pearson and Lipman, Proc. Natl.Acad. Sci. (USA) 85(8), 1988, 2444-2448. A percentage homology oridentity of a homologous polypeptide according to the invention means,in particular, percentage identity of the amino acid residues relativeto the total length of one of the amino acid sequences concretelydescribed herein.

The percentage identity values can also be determined on the basis ofBLAST alignments, the blastp (protein-protein BLAST) algorithm, or usingthe Clustal settings given below.

In the case of a possible protein glycosylation, “functionalequivalents” according to the invention comprise proteins of the typedesignated above in deglycosylated or glycosylated form and modifiedforms obtainable by altering the glycosylation pattern.

Homologs of the proteins or polypeptides according to the invention canbe produced by mutagenesis, e.g. by point mutation, lengthening orshortening of the protein.

Homologs of the proteins according to the invention can be identified byscreening combinatorial libraries of mutants, for example shortenedmutants. For example, a variegated database of protein variants can beproduced by combinatorial mutagenesis at the nucleic acid level, forexample by enzymatic ligation of a mixture of syntheticoligonucleotides. There are a great many methods that can be used forpreparing libraries of potential homologs from a degeneratedoligonucleotide sequence. The chemical synthesis of a degenerated genesequence can be carried out in an automatic DNA synthesizer, and thesynthetic gene can then be ligated into a suitable expression vector.The use of a degenerated set of genes makes it possible to provide allsequences in one mixture, which encode the desired set of potentialprotein sequences. Methods of synthesis of degenerated oligonucleotidesare known by a person skilled in the art (e.g. Narang, S. A. (1983)Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323;Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) NucleicAcids Res. 11: 477).

Several techniques are known in the prior art for screening geneproducts of combinatorial libraries, that have been produced by pointmutations or shortening, and for screening cDNA libraries for geneproducts with a selected property. These techniques can be adapted tothe rapid screening of gene banks that have been produced bycombinatorial mutagenesis of homologs according to the invention. Thetechniques used most often for screening large gene banks, which arebased on a high-throughput analysis, comprise cloning the gene bank intoreplicatable expression vectors, transforming suitable cells with theresultant vector bank and expressing the combinatorial genes underconditions in which detection of the desired activity facilitates theisolation of the vector that encodes the gene whose product wasdetected. Recursive ensemble mutagenesis (REM), a technique thatincreases the frequency of functional mutants in the banks, can be usedin combination with the screening tests, in order to identify homologs(Arkin and Yourvan (1992) PNAS 89: 7811-7815; Delgrave et al. (1993)Protein Engineering 6(3): 327-331).

The invention further comprises the use of the 7β-HSDH wild type fromCollinsella aerofaciens ATCC 25986, as described in the applicant'searlier international patent application PCT/EP2010/068576, which isexpressly referred to hereby.

This 7β-HSDH obtainable from Collinsella aerofaciens DSM 3979 is inparticular characterized by at least one other of the followingproperties, for example 2, 3, 4, 5, 6 or 7 or all such properties:

a) molecular weight (SDS-gel electrophoresis): about 28-32 kDa,especially about 29 to 31 kDa or about 30 kDa;

b) molecular weight (gel filtration, in nondenaturing conditions, suchas in particular without SDS): about 53 to 60 kDa, especially about 55to 57 kDa, such as 56.1 kDa. This proves the dimeric nature of the7β-HSDH from Collinsella aerofaciens DSM 3979;

c) stereoselective reduction of the 7-carbonyl group of 7-keto-LCA to a7β-hydroxyl group;

d) optimal pH for the oxidation of UDCA in the range from pH 8.5 to10.5, especially 9 to 10;

e) optimal pH for the reduction of DHCA and 7-keto-LCA in the range frompH 3.5 to 6.5, especially at pH 4 to 6;

f) at least one kinetic parameter from the following table for at leastone of the substrates/cofactors mentioned there; in the range of 20%,especially 10%, 5%, 3%, 2% or ±1% around the value stated concretely ineach case in the following table.

K_(M) V_(max) k_(cat) (1 μmol/ (μM) (U/mg protein)^(b)) (μmol × min))NADP⁺ 5.32 30.58 944.95 NADPH 4.50 33.44 1033.44 UDCA 6.23 38.17 1179.397-Keto-LCA 5.20 30.77 950.77 DHCA 9.23 28.33 875.35 NAD⁺ —^(a)) — TracesNADH — — Traces ^(a))could not be determined, owing to the very lowactivity ^(b))1 U = 1 μmol/min

g) phylogenetic sequence similarity of the prokaryotic 7β-HSDH fromCollinsella aerofaciens DSM 3979, related to the animal 11β-HSDHsubgroup, comprising Cavia porcellus, Homo sapiens and Mus musculus.

For example, this 7β-HSDH shows the following properties or combinationsof properties: a); b); a) and b); a) and/or b) and c); a) and/or b) andc) and d); a) and/or b) and c) and d) and e); a) and/or b) and c) and d)and e) and f).

A 7β-HSDH of this kind or a functional equivalent derived therefrom ismoreover characterized by

-   a) the stereospecific reduction of a 7-ketosteroid to the    corresponding 7β-hydroxysteroid, and/or-   b) the regiospecific hydroxylation of a ketosteroid comprising a    keto group in 7-position and at least one further keto group on the    steroid skeleton to the corresponding 7β-hydroxysteroid, such as in    particular catalyzed by dehydrocholic acid (DHCA) in 7-position to    the corresponding 3,12-diketo-7β-cholanic acid, and e.g. is    NADPH-dependent.

Said 7β-HSDH has in particular an amino acid sequence according to SEQID NO:2 (accession No.: ZP_01773061) or a sequence derived therefromwith a degree of identity of at least 60%, e.g. at least 65, 70, 75, 80,85, or 90, e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% tothis sequence; optionally additionally characterized by one of thefollowing properties or combinations of properties: a); b); a) and b);a) and/or b) and c); a) and/or b) and c) and d); a) and/or b) and c) andd) and e); a) and/or b) and c) and d) and e) and f) according to theabove definition.

3. Nucleic Acids and Constructs

3. 1 Nucleic Acids

The invention also relates to nucleic acid sequences that code for anenzyme with 7β-HSDH, FDH, GDH and/or 3α-HSDH activity and the mutantsthereof.

The present invention also relates to nucleic acids with a specifieddegree of identity to the concrete sequences described herein.

“Identity” between two nucleic acids means the identity of thenucleotides over the total nucleic acid length in each case, especiallythe identity that is calculated by comparison by means of the Vector NTISuite 7.1 software of the company Informax (USA) employing the Clustalmethod (Higgins D G, Sharp P M. Fast and sensitive multiple sequencealignments on a microcomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1) setting the following parameters:

Multiple alignment parameters: Gap opening penalty 10 Gap extensionpenalty 10 Gap separation penalty range 8 Gap separation penalty off %identity for alignment delay 40 Residue specific gaps off Hydrophilicresidue gap off Transition weighting 0 Pairwise alignment parameters:FAST algorithm on K-tuple size 1 Gap penalty 3 Window size 5 Number ofbest diagonals 5

As an alternative, the identity can also be determined according toChenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson,Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequencealignment with the Clustal series of programs. (2003) Nucleic Acids Res31 (13): 3497-500, according to internet address:http://www.ebi.ac.uk/Tools/clustlw/index.html# and with the followingparameters:

DNA gap open penalty 15.0 DNA gap extension penalty 6.66 DNA matrixIdentity Protein gap open penalty 10.0 Protein gap extension penalty 0.2Protein matrix Gonnet Protein/DNA ENDGAP −1 Protein/DNA GAPDIST 4

All nucleic acid sequences mentioned herein (single- and double-strandedDNA and RNA sequences, for example cDNA and mRNA) can be produced in amanner known per se by chemical synthesis from the nucleotide buildingblocks, for example by fragment condensation of individual overlapping,complementary nucleic acid building blocks of the double helix. Thechemical synthesis of oligonucleotides can be carried out for example ina known manner by the phosphoroamidite method (Voet, Voet, 2nd edition,Wiley Press New York, pages 896-897). The adding on of syntheticoligonucleotides and filling of gaps using the Klenow fragment of DNApolymerase and ligation reactions and general cloning techniques aredescribed in Sambrook et al. (1989), Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press.

The invention also relates to nucleic acid sequences (single- anddouble-stranded DNA and RNA sequences, for example cDNA and mRNA) codingfor one of the above polypeptides and functional equivalents thereof,which are accessible for example using artificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules, whichcode for polypeptides or proteins according to the invention orbiologically active segments thereof, and to nucleic acid fragments thatcan be used e.g. as hybridization probes or primers for identificationor amplification of coding nucleic acids according to the invention.

The nucleic acid molecules according to the invention can moreovercontain untranslated sequences from the 3′- and/or 5′-end of the codinggene region.

The invention further comprises the nucleic acid molecules complementaryto the concretely described nucleotide sequences, or a segment thereof.

The nucleotide sequences according to the invention make it possible toproduce probes and primers that can be used for identification and/orcloning of homologous sequences in other cell types and organisms. Theseprobes or primers usually comprise a nucleotide sequence region, whichhybridizes under “stringent” conditions (see below) to at least about12, preferably at least about 25, for example about 40, 50 or 75successive nucleotides of a sense strand of a nucleic acid sequenceaccording to the invention or a corresponding antisense strand.

An “isolated” nucleic acid molecule is separated from other nucleic acidmolecules that are present in the natural source of the nucleic acid andcan moreover be essentially free from other cellular material or culturemedium, when it is produced by recombinant techniques, or free fromchemical precursors or other chemicals, when it is synthesizedchemically.

A nucleic acid molecule according to the invention can be isolated usingstandard techniques of molecular biology and the sequence informationprovided according to the invention. For example, cDNA can be isolatedfrom a suitable cDNA library, using one of the concretely disclosedcomplete sequences or a segment thereof as hybridization probe andstandard hybridization techniques (as described for example in Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleicacid molecule, comprising one of the disclosed sequences or a segmentthereof, can be isolated by polymerase chain reaction, using theoligonucleotide primers that are prepared on the basis of this sequence.The nucleic acid thus amplified can be cloned into a suitable vector andcan be characterized by DNA sequence analysis. The oligonucleotidesaccording to the invention can also be produced by standard synthesistechniques, e.g. with an automatic DNA synthesizer.

Nucleic acid sequences according to the invention or derivativesthereof, homologs or parts of these sequences can be isolated forexample with usual hybridization methods or PCR technology from otherbacteria, e.g. via genomic or cDNA libraries. These DNA sequenceshybridize under standard conditions to the sequences according to theinvention.

“Hybridize” means the capacity of a polynucleotide or oligonucleotidefor binding to an almost complementary sequence under standardconditions, whereas under these conditions nonspecific bindings do notoccur between noncomplementary partners. For this, the sequences can becomplementary to 90-100%. The property of complementary sequences ofbeing able to bind specifically to one another is utilized for examplein Northern or Southern blotting or in primer binding in PCR or RT-PCR.

Advantageously, short oligonucleotides of the conserved regions are usedfor hybridization. It is also possible, however, to use longer fragmentsof the nucleic acids according to the invention or the completesequences for hybridization. These standard conditions vary depending onthe nucleic acid used (oligonucleotide, longer fragment or completesequence) or depending on which type of nucleic acid, DNA or RNA, isused for the hybridization. Thus, for example, the melting points forDNA:DNA hybrids are approx. 10° C. lower than those of DNA:RNA hybridsof the same length.

Standard conditions mean for example, depending on the nucleic acid,temperatures between 42 and 58° C. in an aqueous buffer solution with aconcentration between 0.1 and 5×SSC (1×SSC=0.15 M NaCl, 15 mM sodiumcitrate, pH 7.2) or additionally in the presence of 50% formamide suchas for example 42° C. in 5×SSC, 50% formamide. Advantageously thehybridization conditions for DNA:DNA hybrids are 0.1×SSC andtemperatures between about 20° C. and 45° C., preferably between about30° C. and 45° C. For DNA:RNA hybrids the hybridization conditions areadvantageously 0.1×SSC and temperatures between about 30° C. and 55° C.,preferably between about 45° C. and 55° C. These temperatures stated forthe hybridization are for example calculated melting point values for anucleic acid with a length of approx. 100 nucleotides and a G+C contentof 50% in the absence of formamide. The experimental conditions for DNAhybridization are described in relevant genetics textbooks, for exampleSambrook et al., “Molecular Cloning”, Cold Spring Harbor Laboratory,1989, and can be calculated from formulas known by a person skilled inthe art for example depending on the length of the nucleic acids, thetype of hybrids or the G+C content. A person skilled in the art can findfurther information on hybridization from the following textbooks:Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, JohnWiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic AcidsHybridization: A Practical Approach, IRL Press at Oxford UniversityPress, Oxford; Brown (ed), 1991, Essential Molecular Biology: APractical Approach, IRL Press at Oxford University Press, Oxford.

“Hybridization” can in particular take place under stringent conditions.These hybridization conditions are described for example in Sambrook,J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (A LaboratoryManual), 2nd edition, Cold Spring Harbor Laboratory Press, 1989, pages9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6.

“Stringent” hybridization conditions are understood in particular as:incubation at 42° C. overnight in a solution consisting of 50%formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodiumphosphate (pH7.6), 5×Denhardt solution, 10% dextran sulfate and 20 g/mldenatured, sheared salmon sperm DNA, followed by a filter washing stepwith 0.1×SSC at 65° C.

The invention also relates to derivatives of the concretely disclosed orderivable nucleic acid sequences.

Thus, further nucleic acid sequences according to the invention can bederived e.g. from SEQ ID NO:1, 5, 7, 14, 19, 34 or 47 and differ fromthem by addition, substitution, insertion or deletion of individual orseveral nucleotides, but furthermore code for polypeptides with thedesired property profile.

The invention also covers those nucleic acid sequences that compriseso-called silent mutations or are altered corresponding to the codonusage of a special original or host organism, compared to a concretelystated sequence, as well as naturally occurring variants, for examplesplice variants or allele variants, thereof.

The invention also relates to sequences obtainable by conservativenucleotide substitutions (i.e. the amino acid in question is replacedwith an amino acid of the same charge, size, polarity and/orsolubility).

The invention also relates to the molecules derived by sequencepolymorphisms from the concretely disclosed nucleic acids. These geneticpolymorphisms can exist between individuals within a population owing tonatural variation. These natural variations usually bring about avariance from 1 to 5% in the nucleotide sequence of a gene.

Derivatives of the nucleic acid sequence according to the invention withthe sequence SEQ ID NO:1, 5, 7, 14, 19, 34 or 47 mean for example allelevariants that have at least 60% homology at the derived amino acidlevel, preferably at least 80% homology, quite especially preferably atleast 90% homology over the total sequence region (regarding homology atthe amino acid level, reference may be made to the above information forthe polypeptides). The homologies can advantageously be higher onpartial regions of the sequences.

Furthermore, derivatives are also to be understood as homologs of thenucleic acid sequences according to the invention, especially of SEQ IDNO:1, 5, 7, 14, 19, 34 or 47, for example fungal or bacterial homologs,shortened sequences, single-stranded DNA or RNA of the coding andnoncoding DNA sequence. For example, homologs to SEQ ID NO:1, 5, 7, 14,19, 34 or 47 possess, at DNA level, a homology of at least 40%,preferably of at least 60%, especially preferably of at least 70%, quiteespecially preferably of at least 80% over the whole DNA region given inSEQ ID NO:1, 5, 7, 14, 19, 34 or 47.

In addition, derivatives are to be understood for example as fusionswith promoters. The promoters that precede the stated nucleotidesequences can be altered by at least one nucleotide exchange, at leastone insertion, inversion and/or deletion, but without the functionalityor effectiveness of the promoters being impaired. Moreover, theeffectiveness of the promoters can be increased by altering theirsequence or they can be exchanged completely with more effectivepromoters even of organisms of a different species.

Furthermore, methods for producing functional mutants are known by aperson skilled in the art.

Depending on the technology used, a person skilled in the art can insertcompletely random or even more targeted mutations in genes or alsononcoding nucleic acid regions (which are for example important for theregulation of expression) and then prepare gene banks. The methods ofmolecular biology required for this are known by a person skilled in theart and for example are described in Sambrook and Russell, MolecularCloning. 3rd edition, Cold Spring Harbor Laboratory Press 2001.

Methods for modifying genes and therefore for modifying the protein thatthese encode have long been familiar to a person skilled in the art,such as for example

-   -   site-directed mutagenesis, giving targeted exchange of        individual or several nucleotides of a gene (Trower M K (Publ.)        1996; In vitro mutagenesis protocols. Humana Press, New Jersey),    -   saturation mutagenesis, in which a codon for any amino acid can        be exchanged or added at any site of a gene (Kegler-Ebo D M,        Docktor C M, DiMaio D (1994) Nucleic Acids Res 22: 1593;        Barettino D, Feigenbutz M, Valcarel R, Stunnenberg H G (1994)        Nucleic Acids Res 22: 541; Barik S (1995) Mol Biotechnol 3: 1),    -   the error-prone polymerase chain reaction (error-prone PCR), in        which nucleotide sequences are mutated by incorrectly        functioning DNA polymerases (Eckert K A, Kunkel T A (1990)        Nucleic Acids Res 18: 3739);    -   the passaging of genes in mutator strains, in which, for example        owing to defective DNA-repair mechanisms, there is an increased        mutation rate of nucleotide sequences (Greener A, Callahan M,        Jerpseth B (1996) An efficient random mutagenesis technique        using an E. coli mutator strain. In: Trower M K (Publ.) In vitro        mutagenesis protocols. Humana Press, New Jersey), or    -   DNA shuffling, in which a pool of closely related genes is        formed and digested and the fragments are used as template for a        polymerase chain reaction, in which through repeated strand        separation and bringing together again, finally mosaic genes of        full length are produced (Stemmer W P C (1994) Nature 370: 389;        Stemmer W P C (1994) Proc Natl Acad Sci USA 91: 10747).

Using so-called directed evolution (described inter alia in Reetz M Tand Jaeger K-E (1999), Topics Curr Chem 200: 31; Zhao H, Moore J C,Volkov A A, Arnold F H (1999), Methods for optimizing industrial enzymesby directed evolution, In: Demain A L, Davies J E (Publ.) Manual ofindustrial microbiology and biotechnology. American Society forMicrobiology), a person skilled in the art can produce functionalmutants in a targeted manner and on a large scale. In a first step,firstly gene banks of the respective proteins are produced, for exampleusing the methods given above. The gene banks are expressed in asuitable manner, for example by bacteria or by phage-display systems.

The relevant genes of host organisms that express functional mutantswith properties that largely correspond to the desired properties can besubmitted to another round of mutation. The steps of mutation and ofselection or screening can be repeated iteratively until the functionalmutants present have the desired properties to a sufficient degree. Withthis iterative procedure, a limited number of mutations, for example 1to 5 mutations, can be performed in steps and assessed and selected fortheir influence on the relevant enzyme property. The selected mutant canthen be submitted to another mutation step in the same way. As a result,the number of individual mutants to be investigated can be reducedsignificantly.

The results according to the invention provide important informationregarding structure and sequence of the enzymes in question, which isnecessary for targeted generation of further enzymes with desiredmodified properties. In particular, so-called “hot spots” can bedefined, i.e. sequence segments that are potentially suitable formodifying an enzyme property by introducing targeted mutations.

3.2 Constructs

The invention further relates to expression constructs containing, underthe genetic control of regulatory nucleic acid sequences, a nucleic acidsequence coding for at least one polypeptide according to the invention;and vectors, comprising at least one of these expression constructs.

“Expression unit” means, according to the invention, a nucleic acid withexpression activity, which comprises a promoter, as defined herein, and,after functional linking with a nucleic acid to be expressed or a gene,regulates the expression, i.e. the transcription and the translation ofthis nucleic acid or of this gene. Therefore the term “regulatorynucleic acid sequence” is also used in this context. In addition to thepromoter, other regulatory elements, for example enhancers, can bepresent.

“Expression cassette” or “expression construct” means, according to theinvention, an expression unit that is functionally linked to the nucleicacid to be expressed or the gene to be expressed. In contrast to anexpression unit, an expression cassette therefore comprises not onlynucleic acid sequences that regulate transcription and translation, butalso the nucleic acid sequences that should be expressed as protein as aresult of the transcription and translation.

The terms “expression” or “overexpression” describe, in the context ofthe invention, the production or increase in the intracellular activityof one or more enzymes in a microorganism, which are encoded by thecorresponding DNA. For this, for example a gene can be inserted in anorganism, a gene that is present can be replaced with another gene, thecopy number of the gene or genes can be increased, a strong promoter canbe used or a gene can be used that codes for a corresponding enzyme witha high activity, and these measures can optionally be combined.

Preferably said constructs according to the invention comprise apromoter 5′-upstream of the respective coding sequence and 3-downstreama terminator sequence and optionally further usual regulatory elements,in each case operatively linked with the coding sequence.

“Promoter”, a “nucleic acid with promoter activity” or a “promotersequence” mean, according to the invention, a nucleic acid, which infunctional linkage with a nucleic acid to be transcribed, regulates thetranscription of said nucleic acid.

“Functional” or “operational” linkage means in this context for examplethe sequential arrangement of one of the nucleic acids with promoteractivity and a nucleic acid sequence to be transcribed and optionallyfurther regulatory elements, for example nucleic acid sequences thatensure the transcription of nucleic acids, and for example a terminator,in such a way that each of the regulatory elements can fulfill itsfunction in the transcription of the nucleic acid sequence. This doesnot necessarily require a direct linkage in the chemical sense. Geneticcontrol sequences, for example enhancer sequences, can exert theirfunction on the target sequence even from more remote positions or evenfrom other DNA molecules. Arrangements are preferred in which thenucleic acid sequence to be transcribed is positioned behind thepromoter sequence (i.e. at the 3′-end), so that the two sequences arelinked together covalently.

The distance between the promoter sequence and the nucleic acid sequencethat is to undergo transgene expression can be less than 200 base pairs,or less than 100 base pairs or less than 50 base pairs.

In addition to promoters and terminators, examples of other regulatoryelements that may be mentioned are targeting sequences, enhancers,polyadenylation signals, selectable markers, amplification signals,replication origins and the like. Suitable regulatory sequences aredescribed for example in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990).

Nucleic acid constructs according to the invention comprise, inparticular, sequence SEQ ID NO:1, 5, 7, 14, 19, 34 or 47 or derivativesand homologs thereof, and the nucleic acid sequences that can be derivedtherefrom, which can advantageously be linked operationally orfunctionally with one or more regulatory signals for controlling, e.g.increasing, gene expression.

In addition to these regulatory sequences, the natural regulation ofthese sequences can still be present before the actual structural genesand optionally can have been genetically modified, so that the naturalregulation is switched off and expression of the genes is increased.However, the nucleic acid construct can also be of simpler construction,i.e. no additional regulatory signals have been inserted before thecoding sequence and the natural promoter with its regulation has notbeen removed. Instead, the natural regulatory sequence is mutated sothat regulation no longer occurs and gene expression is increased.

A preferred nucleic acid construct advantageously also contains one ormore of the aforementioned “enhancer” sequences, functionally linked tothe promoter, which make increased expression of the nucleic acidsequence possible. Additional advantageous sequences can also beinserted at the 3-end of the DNA sequences, such as further regulatoryelements or terminators. The construct can contain one or more copies ofthe nucleic acids according to the invention. The construct can alsocontain further markers, such as antibiotic resistances or auxotrophiccomplementation genes, optionally for selection of the construct.

Examples of suitable regulatory sequences are contained in promoterssuch as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI^(q−), T7,T5, T3, gal, trc, ara, rhaP (rhaP_(BAD))SP6, lambda-P_(R) or in thelambda-P_(L) promoter, which advantageously find application inGram-negative bacteria. Further advantageous regulatory sequences arecontained for example in the Gram-positive promoters amy and SPO2, inthe yeast or fungus promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF,rp28, ADH. Artificial promoters can also be used for regulation.

For expression in a host organism, the nucleic acid construct isadvantageously inserted into a vector, for example a plasmid or a phage,which makes optimal expression of the genes in the host possible. Apartfrom plasmids and phages, vectors are also to be understood as all othervectors known by a person skilled in the art, e.g. viruses, such asSV40, CMV, baculovirus and adenovirus, transposons, IS elements,phasmids, cosmids, and linear or circular DNA. These vectors can bereplicated autonomously in the host organism or can be replicatedchromosomally. These vectors represent another configuration of theinvention.

Suitable plasmids are for example pLG338, pACYC184, pBR322, pUC18,pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24,pLG200, pUR290, pIN-III¹¹³-B1, Agt1 or pBdC in E. coli, pIJ101, pIJ364,pIJ702 or pIJ361 in Streptomyces, pUB110, pC194 or pBD214 in Bacillus,pSA77 or pAJ667 in Corynebacterium, pALS1, pIL2 or pBB116 in fungi,2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 in yeasts or pLGV23, pGHlac⁺,pBIN19, pAK2004 or pDH51 in plants. The aforementioned plasmidsrepresent a small selection of the possible plasmids. Further plasmidsare well known by a person skilled in the art and can for example befound in the book Cloning Vectors (eds. Pouwels P. H. et al. Elsevier,Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In another configuration of the vector, the vector containing thenucleic acid construct according to the invention or the nucleic acidaccording to the invention can also advantageously be inserted in theform of a linear DNA into the microorganisms and can be integrated byheterologous or homologous recombination into the genome of the hostorganism. This linear DNA can consist of a linearized vector such as aplasmid or only of the nucleic acid construct or the nucleic acidaccording to the invention.

For optimal expression of heterologous genes in organisms, it isadvantageous to modify the nucleic acid sequences according to thespecific “codon usage” used in the organism. The “codon usage” caneasily be determined on the basis of computer evaluations of other knowngenes of the organism in question.

An expression cassette according to the invention is prepared by fusionof a suitable promoter with a suitable coding nucleotide sequence and aterminator or polyadenylation signal. For this, usual recombination andcloning techniques are used, such as are described for example in T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)and in T. J. Sihavy, M. L. Berman and L. W. Enquist, Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1984) and in Ausubel, F. M. et al., Current Protocols in MolecularBiology, Greene Publishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acidconstruct or gene construct is advantageously inserted into ahost-specific vector, which makes optimal expression of the genes in thehost possible. Vectors are well known by a person skilled in the art andcan be found for example in “Cloning Vectors” (Pouwels P. H. et al.,Publ., Elsevier, Amsterdam-New York-Oxford, 1985).

4. Microorganisms

Depending on context, the term “microorganism” means the starting(wild-type) microorganism or a genetically modified, recombinantmicroorganism, or both.

Using the vectors according to the invention, recombinant microorganismscan be produced, which for example have been transformed with at leastone vector according to the invention and can be used for producing thepolypeptides according to the invention. Advantageously, the recombinantconstructs according to the invention described above are introducedinto a suitable host system and expressed. Preferably, common cloningand transfection methods known by a person skilled in the art are used,for example coprecipitation, protoplast fusion, electroporation,retroviral transfection and the like, to bring about expression of thestated nucleic acids in the respective expression system. Suitablesystems are described for example in Current Protocols in MolecularBiology, F. Ausubel et al., Publ., Wiley Interscience, New York 1997, orSambrook et al. Molecular Cloning: A Laboratory Manual. 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989. A review of bacterial expression systems forthe heterologous expression of proteins is also provided for example byTerpe, K. Appl. Microbiol. Biotechnol. (2006) 72: 211-222.

In principle, all prokaryotic or eukaryotic organisms may come intoconsideration as recombinant host organisms for the nucleic acidaccording to the invention or the nucleic acid construct.Advantageously, microorganisms such as bacteria, fungi or yeasts areused as host organisms. Advantageously, Gram-positive or Gram-negativebacteria are used, preferably bacteria of the familiesEnterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae orNocardiaceae, especially preferably bacteria of the genera Escherichia,Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella,Agrobacterium, Clostridium or Rhodococcus. The genus and speciesEscherichia coli is quite especially preferred. Further advantageousbacteria can be found, moreover, in the group of alpha-proteobacteria,beta-proteobacteria or gamma-proteobacteria.

The host organism or the host organisms according to the inventionpreferably contain at least one of the nucleic acid sequences, nucleicacid constructs or vectors described in this invention, which code foran enzyme with 7β-HSDH activity according to the above definition.

The organisms used in the process according to the invention are grownor cultured in a manner known by a person skilled in the art, dependingon the host organism. Microorganisms are as a rule grown in a liquidmedium, which contains a carbon source generally in the form of sugars,a nitrogen source generally in the form of organic nitrogen sources suchas yeast extract or salts such as ammonium sulfate, trace elements suchas iron, manganese, magnesium salts and optionally vitamins, attemperatures between 0° C. and 100° C., preferably between 10° C. and60° C. with oxygen aeration. The pH of the liquid nutrient medium can bemaintained at a fixed value, i.e. during growing it may or may not beregulated. Culture can be batchwise, semi-batchwise or continuous.Nutrients can be supplied at the start of fermentation or can bereplenished semi-continuously or continuously.

5. Preparation of UDCA

Step 1: Chemical Reaction from CA to DHCA

The hydroxy groups of CA are oxidized with chromic acid or chromates inacidic solution (e.g. H₂SO₄) to carbonyl groups in a manner known per seby the classical chemical route. DHCA is formed.

Step 2: Enzymatic or Microbial Conversion of DHCA to 12-Keto-UDCA

In aqueous solution, DHCA is reduced by 3α-HSDH and 7β-HSDH or mutantsthereof specifically to 12-keto-UDCA in the presence of NADPH or NADH.The cofactor NADPH or NADH can be regenerated by an ADH or FDH or GDH ormutants thereof from isopropanol or sodium formate or glucose. Thereaction goes under mild conditions. For example, the reaction can becarried out at pH=6 to 9, especially about pH=8 and at about 10 to 30,15 to 25 or about 23° C.

In the case of a microbial reaction step, recombinant microorganismsthat express the necessary enzyme activity/activities can be cultured inthe presence of the substrate to be converted (DHCA) anaerobically oraerobically in suitable liquid media. Suitable cultivation conditionsare known per se by a person skilled in the art. They comprise reactionsin the pH range of for example 5 to 10 or 6 to 9, at temperatures in therange from 10 to 60 or 15 to 45 or 25 to 40 or 37° C. Suitable mediacomprise for example the LB and TB media described below. The reactioncan take place for example batchwise or continuously or in other usualprocess variants (as described above). The reaction time can for examplerange from minutes to several hours or days, and can be e.g. 1 h to 48h. Optionally, if enzyme activity is not expressed continuously, thiscan be induced by adding a suitable inductor, after reaching a targetcell density, e.g. of about OD₆₀₀=0.5 to 1.0.

Further possible suitable modifications of the microbial productionprocess with respect to fermentation mode, additions to the medium,enzyme immobilization and isolation of the valuable substances can alsobe found in the following section concerning “Production of the enzymesor mutants”.

Step 3: Chemical Conversion of 12-Keto-UDCA to UDCA

The 12-carbonyl group of 12-keto-UDCA is removed by means ofWolff-Kishner reduction in a manner known per se, with formation of UDCAfrom 12-keto-UDCA. In the reaction, first the carbonyl group is reactedwith hydrazine to hydrazone. Then the hydrazone is heated in thepresence of a base (e.g. KOH) to 200° C., with cleavage of nitrogen andformation of UDCA.

6. Recombinant Production of the Enzymes and Mutants

The invention further relates to processes for recombinant production ofpolypeptides according to the invention or functional, biologicallyactive fragments thereof, wherein a polypeptide-producing microorganismis cultured, optionally expression of the polypeptides is induced andthe latter are isolated from the culture. The polypeptides can also beproduced on an industrial scale in this way, if this is desirable.

The microorganisms produced according to the invention can be culturedcontinuously or discontinuously in the batch method (batch culture) orin the fed batch or repeated fed batch method. A summary of knowncultivation methods can be found in Chmiel's textbook(Bioprozeßtechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocesstechnology 1. Introduction to bioprocess engineering] (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktorenund periphere Einrichtungen [Bioreactors and peripheral equipment])(Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must suitably fulfill the requirements ofthe respective strains. Descriptions of culture media for variousmicroorganisms are given in the manual “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981).

These media usable according to the invention usually comprise one ormore carbon sources, nitrogen sources, inorganic salts, vitamins and/ortrace elements.

Preferred carbon sources are sugars, such as mono-, di- orpolysaccharides. Very good carbon sources are for example glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch or cellulose. Sugars can also beadded to the media via complex compounds, such as molasses, or otherby-products of sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources areoils and fats such as soybean oil, sunflower oil, peanut oil and coconutoil, fatty acids such as palmitic acid, stearic acid or linoleic acid,alcohols such as glycerol, methanol or ethanol and organic acids such asacetic acid or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials that contain these compounds. Examples of nitrogen sourcescomprise ammonia gas or ammonium salts, such as ammonium sulfate,ammonium chloride, ammonium phosphate, ammonium carbonate or ammoniumnitrate, nitrates, urea, amino acids or complex nitrogen sources, suchas corn-steep liquor, soybean flour, soybean protein, yeast extract,meat extract and others. The nitrogen sources can be used individuallyor as a mixture.

Inorganic salt compounds that can be contained in the media comprise thechloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

The sulfur source used can be inorganic sulfur compounds such assulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfidesbut also organic sulfur compounds, such as mercaptans and thiols.

The phosphorus source used can be phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts.

Chelating agents can be added to the medium in order to keep the metalions in solution. Especially suitable chelating agents comprisedihydroxyphenols, such as catechol or protocatechuate, or organic acids,such as citric acid.

The fermentation media used according to the invention usually alsocontain other growth factors, such as vitamins or growth promoters,which include for example biotin, riboflavin, thiamine, folic acid,nicotinic acid, panthothenate and pyridoxine. Growth factors and saltsare often derived from complex components of the media, such as yeastextract, molasses, corn-steep liquor and the like. Moreover, suitableprecursors can be added to the culture medium. The exact composition ofcompounds in the medium is strongly dependent on the particularexperiment and is decided individually for each specific case.Information on media optimization can be found in the textbook “AppliedMicrobiol. Physiology, A Practical Approach” (Publ. P. M. Rhodes, P. F.Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3). Growth mediacan also be obtained from commercial suppliers, such as Standard 1(Merck) or BHI (brain heart infusion, DIFCO) and the like.

All components of the media are sterilized, either with heat (20 min at1.5 bar and 121° C.) or by sterile filtration. The components can eitherbe sterilized together or separately if necessary. All components of themedia can be present at the start of culture or can optionally be addedcontinuously or batchwise.

The culture temperature is normally between 15° C. and 45° C.,preferably at 25° C. to 40° C. and can be kept constant or varied duringthe experiment. The pH of the medium should be in the range from 5 to8.5, preferably around 7.0. The culture pH can be controlled duringculture by adding basic compounds such as sodium hydroxide, potassiumhydroxide, ammonia or ammonia water or acid compounds such as phosphoricacid or sulfuric acid. To control foaming it is possible to useantifoaming agents, such as fatty acid polyglycol esters. Formaintaining the stability of plasmids, suitable selectively actingsubstances, such as antibiotics, can be added to the medium. To maintainaerobic conditions, oxygen or oxygen-containing gas mixtures, such asambient air, are fed into the culture. The culture temperature isnormally at 20° C. to 45° C. Culture is continued until a maximum of thedesired product has formed. This target is normally reached within 10hours to 160 hours.

The fermentation broth is then processed further. Depending on therequirements, the biomass is removed from the fermentation brothcompletely or partially by separation techniques, such ascentrifugation, filtration, decanting or a combination of these methodsor can be left in it completely.

If the polypeptides are not secreted into the culture medium, the cellscan also be disrupted and the product can be obtained from the lysate byknown methods of protein isolation. The cells can optionally bedisrupted with high-frequency ultrasound, by high pressure, for examplein a French press, by osmolysis, by the action of detergents, lyticenzymes or organic solvents, using homogenizers or by a combination ofseveral of the methods listed.

The polypeptides can be purified by known chromatographic methods, suchas molecular sieve chromatography (gel filtration), such as Q-sepharosechromatography, ion exchange chromatography and hydrophobicchromatography, and with other usual methods such as ultrafiltration,crystallization, salting-out, dialysis and native gel electrophoresis.Suitable methods are described for example in Cooper, F. G.,Biochemische Arbeitsmethoden [Methods of Biochemical Processing], VerlagWalter de Gruyter, Berlin, N.Y. or in Scopes, R., Protein Purification,Springer Verlag, New York, Heidelberg, Berlin.

For isolating the recombinant protein, it may be advantageous to usevector systems or oligonucleotides that lengthen the cDNA with definednucleotide sequences and therefore code for modified polypeptides orfusion proteins, which for example serve for easier purification.Suitable modifications of this kind are for example so-called “tags”functioning as anchors, for example the modification known ashexa-histidine anchors, or epitopes that can be recognized as antigensby antibodies (described for example in Harlow, E. and Lane, D., 1988,Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Theseanchors can serve for attaching the proteins to a solid carrier, forexample a polymer matrix, which can for example be packed in achromatography column, or can be used on a microtiter plate or on someother support.

At the same time, these anchors can also be used for recognizing theproteins. For recognition of the proteins, in addition usual markers,such as fluorescent dyes, enzyme markers, which form a detectablereaction product after reaction with a substrate, or radioactivemarkers, can be used, alone or in combination with the anchors forderivatization of the proteins.

7. Enzyme Immobilization

In the method described herein, the enzymes according to the inventioncan be used free or immobilized. An immobilized enzyme is to beunderstood as an enzyme that is fixed to an inert support. Suitablesupport materials and the enzymes immobilized thereon are known fromEP-A-1149849, EP-A-1 069 183 and DE-OS 100193773 and from the literaturereferences cited therein. Regarding this, full reference is made to thedisclosure of these documents. Suitable support materials include forexample clays, clay minerals, such as kaolinite, diatomaceous earth,perlite, silicon dioxide, aluminum oxide, sodium carbonate, calciumcarbonate, cellulose powder, anion exchanger materials, syntheticpolymers, such as polystyrene, acrylic resins, phenol-formaldehyderesins, polyurethanes and polyolefins, such as polyethylene andpolypropylene. For preparing the supported enzymes, the supportmaterials are usually used in a finely-divided, particulate form, withporous forms being preferred. The particle size of the support materialis usually not more than 5 mm, especially not more than 2 mm(particle-size distribution curve). Similarly, when using dehydrogenaseas whole-cell catalyst, a free or immobilized form can be selected.Support materials are for example Ca-alginate, and carrageenan. Enzymesas well as cells can also be crosslinked directly with glutaraldehyde(crosslinking to CLEAs). Corresponding and further methods ofimmobilization are described for example in J. Lalonde and A. Margolin“Immobilization of Enzymes” and in K. Drauz and H. Waldmann, EnzymeCatalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH,Weinheim.

Experimental Section

Unless stated otherwise, the cloning steps carried out in the context ofthe present invention, for example restriction cleavage, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids onto nitrocellulose and nylon membranes, linkage of DNA fragments,transformation of microorganisms, culturing of microorganisms,multiplication of phages and sequence analysis of recombinant DNA arecarried out as described in Sambrook et al. (1989) op. cit.

A. General Information

Materials:

The genomic DNA of Collinsella aerofaciens DSM 3979 (ATCC 25986, formerdesignation Eubacterium aerofaciens) was obtained from the GermanCollection of Microorganisms and Cell Cultures (DSMZ). UDCA and7-keto-LCA are starting compounds that are known per se and aredescribed in the literature. All other chemicals were obtained fromSigma-Aldrich and Fluka (Germany). All restriction endonucleases, T4 DNAligase, Taq DNA polymerase, Phusion DNA polymerase andisopropyl-β-D-1-thiogalactopyranoside (IPTG) were obtained fromFermentas (Germany).

Media:

LB medium, containing tryptone 10 g, yeast extract 5 g, NaCl 10 g perliter of medium

TB medium, containing tryptone 12 g, yeast extract 24 g, 4 mL glycerol,10% TB buffer (11.55 g KH₂PO₄, 62.7 g K₂HPO₄, H₂O to 500 mL) per literof medium

Minimal medium, modified according to Wilms et al., BIOTECHNOLOGY ANDBIOENGINEERING, 2001 VOL. 73, No. 2, 95-103

Expression Vectors and Vector Constructs

For the expression of recombinant proteins, the following expressionvectors that are known per were used:

pET21a(+), (cf. FIG. 3a )

pET22b(+) (cf. FIG. 3b )

pET28a(+) (cf. FIG. 3d ) and

pCOLADuet-1

(in each case Novagen, Madison, Wis., USA).

The vectors pET21a(+) and pET22b(+) each possess a multiple cloning site(MCS), in each case under the control of a T7-promoter with downstreamlac-operator and the subsequent ribosomal binding site (rbs). In theC-terminal region of the expression domain there is in each case aT7-terminator. Both plasmids have a ColE1-replicon (pBR322-replicon), anampicillin resistance gene (b/a), an f1-origin and a gene coding for thelac-inhibitor (lacI).

In addition, the pET21a(+)-plasmid has a T7-Tag in the N-terminal regionof the MCS and an optional His-Tag C-terminally of the MCS. ThepET22b(+)-plasmid has, in the N-terminal region of the MCS, a pelBsignal sequence and a His-Tag C-terminally of the MCS.

The pCOLADuet-1 vector has two MCSs, each of which is under the controlof a T7-promoter with downstream lac-operator and subsequent ribosomalbinding site (rbs). C-terminally of the two MCSs there is aT7-terminator. Moreover, this vector has a gene that codes for thelac-inhibitor and a COLA-replicon (ColA-replicon).

A modified variant of the commercially available pCOLADuet-1 vector wasused in the present work. In this modified plasmid variant (designatedpCOLA(mod); cf. FIG. 3c )) the kanamycin resistance gene of the originalvector was replaced with a chloramphenicol resistance gene. In addition,an NcoI restriction site was removed from the chloramphenicol resistancegene by site-directed point mutagenesis. In the N-terminal region of thefirst MCS there is a His-Tag, whereas C-terminally of the second MCSthere is an S-Tag.

The ColE1-replicons of the pET-plasmids and the COLA-replicon of thepCOLA-plasmid are mutually compatible. This permits simultaneous stableinsertion of a pET-plasmid and of a pCOLA-plasmid in Escherichia coli.In this way, combinations of various genes can be cloned intoEscherichia coli, without their being located on the same operon. Owingto the different copy numbers of pET-vectors (˜40) and pCOLA-vectors(20-40), it is moreover possible to influence the expression level ofcotransformed genes.

The following vector constructs were used

pET22b(+) 7β-HSDH: a pET22b(+) vector into which the 7β-HSDH fromCollinsella aerofaciens ATCC 25986 had been cloned via the Nde I andHind III cleavage sites in the usual way.

pET22b(+) 3α-HSDH: a pET22b(+) vector into which the 3α-HSDH fromComamonas testosteroni had been cloned via the Nde I and EcoR I cleavagesites in the usual way (Oppermann et al., J Biochem, 1996, 241(3):744-749).

pET21a(+) FDH D221G (cf. FIG. 4a ): a pET21a(+) vector into which theformate dehydrogenase from Mycobacterium vaccae N10 had been cloned viathe Nde I and EcoR I cleavage sites. With site-directed mutagenesis, theaspartate residue (D) at position 221 (without taking into accountmethionine in position 1) or position 222 (counting from methionine inposition 1; cf. SEQ ID NO: 15, 19, 35) of formate dehydrogenase wasreplaced with a glycine residue (cf. production example 4, below).Formate dehydrogenase carries, at position 1202 of the nucleotidesequence, a single base deletion, which leads to exchange of the lastamino acid valine for an alanine. Simultaneously, this base deletionleads to switching-off of the stop codon and to activation of theHis-Tag that was originally outside of the reading frame (cf. SEQ ID NO:34 and 35).

pET21a(+) 7β-HSDH: a pET21a(+) vector, into which the 7β-HSDH fromCollinsella aerofaciens ATCC 25986 had been cloned via the Nde I and XhoI cleavage sites in the usual way

pET21a(+) FDH D221G 7β-HSDH (cf. FIG. 4b )

pET21a(+) FDH 7β-HSDH(G39A) 3α-HSDH (cf. FIG. 8)

pCOLA(mod) 3α-HSDH (cf. FIG. 5)

Microorganisms

Strain Genotype Escherichia coli DH5α F⁻ endA1 glnV44 thi-1 recA1 relA1gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA- argF)U169, hsdR17(r_(K) ⁻m_(K)⁺),λ− Escherichia coli F⁻ ompT gal dcm Ion hsdS_(B)(r_(B) ⁻m_(B) ⁻)λ(DE3 BL21(DE3) [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) Escherichia coliBL21 F⁻ ompT gal dcm Ion hsdS_(B)(r_(B) ⁻m_(B) ⁻) λ(DE3 (DE3) hdhA⁻KanR⁺ [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) (7α-HSDH knock-out hdhA⁻KanR⁺ strain) (cf. production example 5)

The Escherichia coli strain DH5α (Novagen, Madison, Wis., USA) wasmultiplied at 37° C. in LB medium containing suitable antibiotics.

The Escherichia coli strain BL21(DE3) (Novagen, Madison, Wis., USA) wasmultiplied at 37° C. in LB medium containing suitable antibiotics andafter induction at OD₆₀₀=0.8 with 0.5 mM IPTG was held at 25° C. and 140rpm.

Methods

1. Standard Conditions for Determination of 7β-HSDH Activity

The reaction mixture contains a total volume of 1 ml:

880 μl 50 mM potassium phosphate buffer, pH 8.0

10 μl 10 mM UDCA (dissolved in water, pH 8)

10 μl enzyme solution (in buffer as above, in the range from 1 to 10U/ml)

100 μl 1 mM NADP⁺ (in buffer as above)

The increase in extinction at 340 nm is measured and the activity iscalculated as enzyme unit (U, i.e. μmol/min) using the molar extinctioncoefficient of 6.22 mM⁻¹×cm⁻¹.

Standard Conditions for Determination of 7β-HSDH Activity According toProduction Example 7

The reaction mixture contains a total volume of 1 ml

870 μl 50 mM potassium phosphate (KPi) buffer, pH 8.0

100 μl 100 mM DHCA (dissolved in 50 mM KPi, pH 8)

10 μl enzyme solution (in buffer as above, in the range from 2 to 6U/ml)

20 μl 12.5 mM NADPH (dissolved in ddH₂O)

The increase in extinction at 340 nm is measured and the activity iscalculated as enzyme unit (U, i.e. μmol/min) using the molar extinctioncoefficient of 6.22 mM⁻¹×cm⁻¹.

2. Determination of Protein by BCA Assay

The samples were mixed with BCA reagent (from Interchim) and incubatedat 37° C. for 45 min. The protein content was determined at 562 nmagainst a calibration curve (BSA) in the concentration range of theassay used.

3. Thin-Layer Chromatography

5 to 10 μg of sample was applied to a TLC Film Silica Gel 60 (Merck).Authentic substances were applied as reference. One end of the TLC filmwas dipped in solvent until the top of the mobile phase was reached. TheTLC film was dried and was developed with phosphomolybdic acid.

4. Molecular Biology Procedures:

Molecular biology operations are carried out, unless stated otherwise,on the basis of established methods, e.g. described in:

-   Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A    Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold    Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989;    Ausubel et al. (eds.), Current Protocols in Molecular Biology, John    Wiley & Sons, N Y (1993); Kriegler, Gene Transfer and Expression, A    Laboratory Manual, Stockton Press, NY (1990).

B. EXAMPLES Production Example 1: Identification of 7β-HSDH Activity

The genomic DNA sequence of Collinsella aerofaciens ATCC 25986 waspublished in the year 2007 by “Washington University Genome SequencingCenter” for the “human gut microbiome project” at GenBank. HSDHs belongto the “short-chain dehydrogenases”. As the biochemical function of the“short-chain dehydrogenases” from Collinsella aerofaciens ATCC 25986 hadnot been annotated in GenBank, 9 candidates were cloned into vectorpET22b+, and then expressed in E. coli BL21(DE3).

For this, 7β-HSDH coding sequences were PCR-amplified. The PCR productswere obtained using the genomic DNA of Collinsella aerofaciens ATCC25986 (DSM 3979) as template and the primers5′-gggaattcCATATGAACCTGAGGGAGAAGTA-3′ (SEQ ID NO:3) and5′-cccAAGCTTCTAGTCGCGGTAGAACGA-3′ (SEQ ID NO:4). The NdeI and HindIIIcleavage sites in the primer sequences are underlined. The PCR productwas purified using the PCR-Purification-Kit (Qiagen) and then cut withthe enzymes NdeI and HindIII. The corresponding vector was also cut withNdeI and HindIII. The products were applied to agarose gel, separated,cut out and purified. The cut PCR product and the cut vector wereligated by means of T4-ligase. The ligant was transformed into E. coliDH5a. The resultant vector (contains the gene of 7β-HSDH) was confirmedby sequencing and transformed into E. coli BL21(DE3) and induced withIPTG and expressed.

Expression was carried out in 50 ml LB medium. For preparation of apreculture, a colony on LB-agar plate in 5 ml LB medium (containscorresponding antibiotics) was picked and incubated overnight at 37° C.and 160 rpm. The 50 ml LB medium (contains corresponding antibiotics)was inoculated with 500 μl of preculture. The culture was incubated at37° C. and 160 rpm. Up to OD600 approx. 0.8, expression was induced byadding 0.5 mM IPTG. After 6 h or overnight, the cells were centrifugedoff. The pellets were resuspended in 6 ml potassium phosphate buffer (50mM, pH 8, contains 0.1 mM PMSF) and disrupted with ultrasound. The celldebris was removed by centrifugation.

For identification of 7β-HSDH activity, the activity was investigated bya photometric method. In a 1 ml cuvette, enzyme and 0.1 mM of testsubstance (UDCA) were mixed in potassium phosphate buffer (50 mM, pH8).After adding NADPH(NADH) or NADP⁺(NAD⁺), the degradation or formation ofNAD(P)H was measured. One enzyme out of the 9 candidates shows activity(60 U/ml) against UDCA in the presence of NADP⁺, but no activity againstCA. The NADPH-dependent 7β-HSDH activity of this enzyme was identified.

In the photometric investigation, 7β-HSDH showed activity of 60 U/mlagainst UDCA, activity of 35 U/ml against 7-keto-LCA and activity of 119U/ml against DHCA in the presence of NADP⁺ or NADPH. The activityagainst CA is not detectable.

The gene that codes for 7β-HSDH was subcloned into pET28a+ with His-Tag,to permit rapid purification. This 7β-HSDH with His-Tag was activelyexpressed in E. coli BL21(DE3) as described above. Purification wascarried out with a Talon column. The column was first equilibrated withpotassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl). Afterloading of the cell lysate, the column was washed with potassiumphosphate buffer (50 mM, pH 8, with 300 mM NaCl). The 7β-HSDH was elutedwith potassium phosphate buffer (50 mM, pH 8, with 300 mM NaCl and 200mM imidazole). The imidazole in the eluate was removed by dialysis. Theyield in purification was 76% with purity of approx. 90%.

Production Example 2: Preparative-Scale Cloning, Expression andPurification of 7β-HSDH from Collinsella aerofaciens ATCC 25986 andFurther Characterization of the Enzyme

2.1 Cloning and Production of an Expression Construct

The gene coding for 7β-HSDH was once again amplified from the genomicDNA by PCR and using primers, as described above for production example1:

The PCR product was once again purified as described above and digestedwith the restriction endonucleases NdeI and HindIII. The digested PCRproduct was purified again and cloned into the pET-28a(+) vector usingthe T4-ligase, to produce an expression vector. The resultant expressionconstruct was then transformed into E. coli DH5α cells. The protein tobe expected should have 20 amino acid residues comprising a signalpeptide and an N-terminal 6×His-Tag and a thrombin cleavage site. Thesequence of the inserted DNA was verified by sequencing.

2.2 Overexpression and Purification of 7β-HSDH

E. coli BL21(DE3) was transformed with the expression construct. Forthis, the E. coli BL21(DE3) strain containing the expression constructwas multiplied in LB medium (2×400 ml in 2-liter shaking bottles)containing 30 μg/ml kanamycin. The cells were harvested bycentrifugation (10.000×g, 15 min, 4° C.). The pellet was resuspended in20 ml phosphate buffer (50 mM, pH 8, containing 0.1 mM PMSF). The cellswere disrupted by ultrasound treatment for 1 minute with constantcooling (40 W power, 40% working interval and 1 min pause) usingSonifier 250 ultrasonic equipment (Branson, Germany). Lysis was repeatedthree times. The cell extract was centrifuged (22.000×g, 20 min, 4° C.).The supernatant was loaded on a Talon column (Clontech, USA),equilibrated with loading buffer (50 mM potassium phosphate, 300 mMNaCl, pH 8). The procedure was carried out at 24° C. Unbound materialwas washed away by washing the column with loading buffer (3 columnvolumes). Weakly binding protein was removed by washing with washingbuffer (20 mM imidazole in the loading buffer; 3 column volumes). TheHis-Tag-7β-HSDH protein was eluted with elution buffer (200 mM imidazolein the loading buffer). The eluate was dialyzed overnight in a dialysistube with a molecular exclusion limit of 5 kDa (Sigma, USA) in 2 litersof potassium phosphate buffer (50 mM, pH 8) at 4° C. Finally the samplewas transferred to a new tube and stored at −20° C. for furtheranalysis. The protein concentration was determined using a BCA Test Kit(Thermo, USA) according to the manufacturer's instructions. In additionthe sample was analyzed by 12.5% SDS-PAGE and staining with CoomassieBrilliant Blue. The purity of the protein was determineddensitometrically using Scion Image Beta 4.0.2 (Scion, USA).

2.3 Gel Filtration

Gel filtration was carried out on a Pharmacia AKTA protein purificationsystem, in order to determine the molecular weight of 7β-HSDH. Thepurified enzyme was applied on a Sephadex G-200 column, which had beenequilibrated beforehand with 50 mM Tris-HCl (pH 8), containing 200 mMsodium chloride. The protein was eluted with the same buffer at a flowrate of 1 ml/min. The molecular weight of 7β-HSDH was determined bycomparing its elution volume with that of protein standards (serumalbumin (66 kDa), α-amylase from Aspergillus oryzae (52 kDa), pigpancreas trypsin (24 kDa) and hen's egg lysozyme (14.4 kDa)).

2.4 Enzyme Assay and Kinetic Analysis

The reaction mixture for the enzyme assay contained, in a total volumeof 1 ml, 50 μmol potassium phosphate (pH 8), 0.1 μmol NAD(P)H orNAD(P)⁺, substrates and protein. The reaction mixture was contained incuvettes with a light path length of 1 cm. The 7β-HSDH activity wasdetermined by recording the variation in NAD(P)H concentration againstthe extinction at 340 nm using a spectrophotometer (Ultraspec 3000,Pharmacia Biotech, Great Britain). The enzyme activities were determinedas enzyme units (U, i.e. μmol/min) using the molar extinctioncoefficient of 6.22 mM⁻¹−cm⁻¹ at 25° C. Several different measurementswere performed with the variables substrate, coenzyme, concentration,pH, buffer and incubation temperature. The kinetic constants weredetermined using standard methods.

2.5 Biotransformation of 7-Keto-Lithocholic Acid by 7β-HSDH

The transformation of 7-keto-LCA by 7β-HSDH was carried out in order toverify the biochemical function of 7β-HSDH. 0.4 g 7-keto-LCA wassuspended in 10 ml potassium phosphate buffer (50 mM, pH 8) and the pHwas adjusted to pH 8 by adding 2 M sodium hydroxide. 0.2 ml isopropanol,100 U 7β-HSDH and 80 U alcohol dehydrogenase (ADH-TE) fromThermoanaerobacter ethanoicus (kindly donated by Dr. K. Momoi, ITBUniversity, Stuttgart) and 1 μmol NADP⁺ were added. The same buffer wasadded, to give a total reaction volume of 20 ml. The reaction mixturewas incubated at 24° C. and stirred for 24 hours. During this, NADPH wasregenerated with ADH via oxidation of 2-propanol. The product wasacidified with 1 ml of 2 M hydrochloric acid and was extracted 5× with 5ml ethyl acetate. The organic solution was then distilled.

2.6 Determination of Chromatographic Product

An HPLC analysis was carried out on a column of the Purospher® STARRP-18 type (Hitbar® RT 125-4 Pre-Packed Column, Purospher® STAR RP-18endcapped, Merck, Germany), provided with a precolumn of the LiChroCART®STAR RP18 type (endcapped, Merck, Germany) on an HPLC system LC20AD(Shimadzu, Japan) at a flow rate of 1 ml/min. The mobile phase consistedof two eluents. Eluent A contained acetonitrile and eluent B containeddistilled water (pH 2.6, adjusted with orthophosphoric acid, 85%). Thefollowing gradient was used: A 35% (8 min)-35%-43% (1% min⁻¹)-43%-70%(1% min⁻¹)-70% (5 min)-70%-35% (17.5% min⁻¹)-35% (5 min); eluent A 65%(8 min)-65%-57% (1% min⁻¹)-57%-30% (1% min⁻¹)-30% (5 min)-30%-65% (17.5%min⁻¹)-65% (5 min). 20 μl samples (1 mg/ml) were analyzed. AuthenticUDCA, 7-keto-LCA and CDCA were used at the same concentration asstandards. Recording was carried out by UV detection at 200 nm.

2.7 Sequence Alignment and Phylogenetic Analysis

Multiple sequence alignments were produced using the Clustal X-Software(Thompson et al., 1997, Nucleic Acid Research 25: 4876-82) and modifiedusing the Jalview software (Clamp et al., 2004, Bioinformatics 20:426-7). The phylogenetic tree was produced using the program TreeView1.6.6 (Roderic 2001, http://taxonomy.zoology.gla.ac.kuk/rod/rod.html).

2.8 Test Results:

2.8.1 Identification of 7β-HSDH Activity in a PreparativeBiotransformation

To confirm the function of the enzyme, a biotransformation of 7-keto-LCAwas carried out at the 10-ml scale, wherein the isolated enzyme was usedin combination with ADH for regeneration of NADPH using 2-propanol, asalready described. The HPLC analysis showed that UDCA was the onlyreaction product produced by the enzyme (90% conversion). CDCA(retention time 19.4 min) was not detected in the reaction mixture. Theresult shows that the enzyme is an NADPH-dependent 7β-HSDH and iscapable of selective reduction of the 7-carbonyl group of 7-keto-LCA toa 7β-hydroxyl group.

Retention time UDCA: 15.5 min

Retention time 7-keto-LCA: 18.3 min

2.8.2. Purification and Gel Filtration

After cloning the 7β-HSDH gene from Collinsella aerofaciens DSM 3979into the expression vector pET28a(+) and subsequent overexpression, afusion protein provided with a His-Tag on the N-terminus was obtainedwith a 7β-HSDH yield of 332.5 mg (5828 U) per liter of culture. The7β-HSDH provided with the His-Tag was purified in one step byimmobilized metal ion affinity chromatography (purity>90%, yield 76%,cf. FIG. 2). The main bands of lanes 1 and 2 represent the expectedexpression product at 30 kDa, which corresponds to the predictedmolecular weight derived from the amino acid sequence of the gene.However, a molecular weight of 56.1 kDa is found for 7β-HSDH by gelfiltration. This proves the dimeric nature of the 7β-HSDH fromCollinsella aerofaciens DSM 3979.

2.8.3. Sequence Alignments

The amino acid sequence of the 7β-HSDH used according to the inventionwas compared with known HSDH sequences (alignment not shown). Theobserved sequence similarity indicates that the enzyme according to theinvention belongs to the family of short-chain dehydrogenases (SDR). Itis known that SDRs have very low homology and sequence identity(Jornvall, H., B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J.Jeffery, and D. Ghosh. 1995. Short-chain dehydrogenases/reductases(SDR). Biochemistry 34: 6003-13 and Persson, B., M. Krook, and H.Jornvall. 1991. Characteristics of short-chain alcohol dehydrogenasesand related enzymes. Eur J Biochem 200: 537-43). However, the sequencealignment clearly shows the conserved domains in the SDR primarystructure. The N-terminal motif Gly-X-X-X-Gly-X-Gly (corresponding toGly-41, Gly-45 and Gly-47, numbering corresponding to the alignment)corresponds to the characteristic dinucleotide binding motif of the SDRsuperfamily. Furthermore, three strongly conserved residues Ser-177,Tri-190 and Lys-194 are observed, which correspond to the catalytictriad of the SDR enzymes.

2.8.4. Phylogenetic Analysis

7α-HSDHs from Clostridium sordellii, Brucella melitensis and Escherichiacoli belong to the same subgroup. The two 3α-HSDHs show a morepronounced similarity than other HSDHs. Interestingly, the prokaryotic7β-HSDH is related to the animal 11β-HSDH subgroup, comprising Caviaporcellus, Homo sapiens and Mus musculus.

2.8.5. Kinetic Constants

Kinetic equilibrium analyses were carried out, in order to determine theabsolute values for V_(max) and K_(M) for UDCA, 7-keto-LCA, DHCA, NADP⁺and NADPH by Lineweaver-Burk plots. The following table presents allkinetic data for the substrates and coenzymes tested, which wereobtained from substrate saturation curves and reciprocal plots. TheV_(max), K_(M) and k_(cat) values for all substrates and coenzymes arein the same region, whereas the K_(M) value for DHCA was significantlyhigher than with the other substrates, possibly caused by the lowsolubility in water. The enzyme is NADPH-dependent and kinetic constantscould not be determined for NAD⁺ and NADH owing to the very lowactivity.

Summary of kinetic constants for 7β-HSDH from Collinsella aerofaciensDSM 3979.

K_(M) V_(max) k_(cat) (1 μmol/ (μM) (U/mg protein)^(b)) (μmol × min))NADP⁺ 5.32 30.58 944.95 NADPH 4.50 33.44 1033.44 UDCA 6.23 38.17 1179.397-Keto-LCA 5.20 30.77 950.77 DHCA 9.23 28.33 875.35 NAD⁺ —^(a)) — TracesNADH — — Traces ^(a))could not be determined owing to the very lowactivity ^(b))1 U = 1 μmol/min2.8.6. Optimal pH

In addition, the 7β-HSDH activity was determined with purified enzymefor various substrates as a function of the pH. For the oxidation ofUDCA with 7β-HSDH, an optimal activity was observed in the range from pH9 to 10 with gradual decline on the acidic side. In contrast, for thereduction of DHCA and 7-keto-LCA by 7β-HSDH, there was an optimalactivity in the range from pH 4 to 6 with a sharp decline on the acidicside and a gradual decline on the alkaline side. Different buffers onlyhave a slight influence on the activity of 7β-HSDH at identical pH.

2.8.7. Thermal Stability

The NADP-dependent 7β-HSDH used according to the invention shows thefollowing stability behavior: after 400 min the activity at 30° C. wasabout 30% lower than at 23° C. At 30° C., the enzyme was completelyinactivated after 1500 min, whereas at 23° C. and 1500 min the residualactivity was 20%. No significant activity loss was observed duringstorage at −20° C. in potassium phosphate buffer (50 mM, pH 8) over aperiod of some months after repeated freezing and thawing.

Production Example 3: Production of 7β-HSDH Mutants and CharacterizationThereof

Position 39 of the amino acid sequence (comprising start methionine)(cf. SEQ ID NO:2) was mutated.

3.1 Primers

The mutagenesis primers stated below were used for the site-directedmutagenesis of 7β-HSDH. The primers were selected based on the 7β-HSDHgene sequence, so that they bring about the desired amino acid exchange.It was borne in mind that the base to be mutated is localized centrallyin the primer, and that the melting points of the primer pairs are inthe same region.

The primer pair 7beta_mut_G39A_fwd and 7beta_mut_G39A_rev was used forpreparing the G39A mutant. The primer pair 7beta_mut_G39S_fwd and7beta_mut_G39S_rev was used for preparing the G39S mutant.

Glycine→Alanine Forward:  7beta_mut_G39A_fwd:  SEQ ID NO: 9)CGTCGTCATGGTCGCCCGTCGCGAGG. Reverse:  7beta_mut_G39A_rev: SEQ ID NO: 10)CCTCGCGACGGGCGACCATGACGACG. Glycine→Serine Forward:  7beta_mut_G39S_fwd:SEQ ID NO: 11) CGTCGTCATGGTCAGCCGTCGCGAGG. Reverse:  7beta_mut_G39S_rev:SEQ ID NO: 12) CCTCGCGACGGCTGACCATGACGACG.3.2 PCR Program

In the reaction, first a 2-min initial denaturation step was carried outat 98° C. Then 25 cycles of denaturation (30 s at 98° C.), primerhybridization (2.5 min at 58° C.) and elongation (6 min at 72° C.) werecarried out. As the last step, a final elongation of 15 min was carriedout at 72° C. before the polymerase chain reaction was stopped bycooling to 4° C.

3.3 PCR assay

HF buffer (5x) 4 μl dNTP-mix (10 mM) 0.4 μl Forward primer (10 μM) 2 μlReverse primer (10 μM) 2 μl Template 1 μl Phusion polymerase (2 U μL⁻¹)0.2 μl DMSO 1 μl ddH₂O 9.4 μl 20 μl A pET22b vector with 7β-HSDH wasused as template.3.4 Procedure

To allow targeted exchange of amino acids in protein sequences, the DNAsequence of the corresponding gene is submitted to site-directedmutation. For this, mutually complementary primers are used, which bearthe desired mutation in their sequence. N6-adenine-methylated,double-stranded plasmid DNA, which bears the gene to be mutated, servesas template. N6-adenine-methylated plasmid DNA is isolated from dam⁺ E.coli strain such as for example E. coli DH5.

The polymerase chain reaction is carried out as described above. Theprimers are lengthened complementarily to the template, so that plasmidswith the desired mutation are formed, which have a strand break. Unlikeother PCR reactions, in this case the increase in DNA yield is onlylinear, as newly formed DNA molecules cannot serve as template for thePCR reaction.

On completion of the PCR reaction, the parental, N6-adenine-methylatedDNA is digested by the restriction enzyme DpnI. This enzyme has theparticular feature that it restricts nonspecificallyN6-adenine-methylated DNA, but not the newly formed, nonmethylated DNA.Restriction was carried out by adding 1 μL DpnI to the PCR reactionmixture and incubating for 1 h at 37° C.

10 μl of this preparation was used for the transformation of 200 μl ofchemically-competent DH5α cells. After plasmid isolation, successfulmutation was confirmed by sequencing.

3.5 Characterization

For each of the mutants and for the wild-type enzyme, kineticmeasurements were carried out in the microtiter plate photometer,recording the conversion rates of the enzymes both with constantsubstrate concentrations with variation of cofactor concentrations andvice versa. To determine the specific enzyme activity, the enzymeconcentrations in the cell lysate were determined by densitometry.

To determine the dependence of the substrate conversion rate on thesubstrate concentration, a cofactor concentration of 100 μM NADPHrelative to the reaction volume was used, while the substrateconcentration was varied over a range from 7 μM to 10 mM dehydrocholicacid with 31 different concentrations (in each case relative to thereaction mixture).

To determine the dependence of the substrate conversion rate on thecofactor concentration, a substrate concentration of 0.3 mMdehydrocholic acid relative to the reaction volume was used, whereas thecofactor concentration was varied over a range from 25 μM to 100 μMNADPH with 8 different concentrations of the wild-type protein or over arange from 6 μM to 100 μM NADPH with 16 different concentrations forboth mutants (in each case relative to the reaction mixture). In thesemeasurement series, the reaction rate was determined by linearregression over the first 4 measurements (0 s-18 s).

The data obtained were evaluated with IGOR Pro. From the plots ofsubstrate or cofactor concentration against the specific enzymeactivity, the typical curve of Michaelis-Menten kinetics can be seen forthe measurement series at constant substrate concentration and variationof cofactor concentration. From the plots of the measurement series withconstant cofactor concentration and variation of substrateconcentration, however, typical curves of Michaelis-Menten kinetics withsubstrate inhibition can be seen (cf. FIG. 6). For this reason, theclassical Michaelis-Menten model was used for evaluating the measurementseries at constant substrate concentration and variation of cosubstrateconcentration, and the Michaelis-Menten model with substrate inhibitionwas used for the measurement series with constant cosubstrateconcentration and variation of substrate concentration.

v_(max), K_(m) and K_(i) were determined by nonlinear regression.

$v = {v_{\max} \cdot \frac{c_{S}}{K_{m} + {\left( {1 + \frac{c_{S}}{K_{i}}} \right)c_{S}}}}$v specific enzyme activity, U mg⁻¹=μmol min⁻¹ mg⁻¹v_(max) maximum specific enzyme activity, U mg⁻¹=μmol min⁻¹ mg⁻¹c_(s) substrate or cofactor concentration, mol L⁻¹K_(m) semisaturation concentration, mol L⁻¹K_(i) inhibition constant, mol L⁻¹The parameters found are shown in the table.

TABLE Parameters found for the three different 7β-HSDH variantsK_(m, DCS), K_(i, DCS), K_(m, NADPH), v_(max), μM mM μM U/mg 7β-HSDH WT31 ± 5 8.6 ± 1.5 46 ± 7 14.6 ± 1.0 7β-HSDH G39A 33 ± 6 17 ± 4  16.4 ±1.9 21.0 ± 1.1 7β-HSDH G39S 75 ± 9 80 ± 50 13.1 ± 2.9 20.7 ± 1.0

On examining the measured data, it is clear that 7β-HSDH displayssubstrate inhibition via dehydrocholic acid, which is strongest for thewild-type protein and weakest with the G39S mutant. Especially in thecase of this last-mentioned protein, it may be asked whether substrateinhibition occurs at all, as indicated by the high K_(i) and its largeerror. The semisaturation concentrations for dehydrocholic acid are inthe two-digit micromolar range. Whereas these do not differsignificantly for the wild-type protein and the G39A mutant, at 31±5 μMand 33±6 μM respectively, for the G39S mutant this value, at 75±9 μM, isroughly double the value for the other two enzymes. In the case of thesemisaturation concentrations for NADPH, lower values are found for themutant enzymes than for the wild-type enzyme (46±7 μM in the wild-typeversus 16.4±1.9 μM for the G39A mutant and 13.1±2.9 μM for the G39Smutant).

For both mutants, the values for v_(max) are roughly 1.5 times the valueof the wild-type protein (21.01.1 U/mg for the G39A mutant and 20.1±1.0U/mg for the G38S mutant versus 14.6±1.0 U/mg for the wild-typeprotein). In contrast to the classical Michaelis-Menten model, however,for the Michaelis-Menten model with substrate inhibition the v_(max)values are not to be regarded as maximum attainable specific enzymeactivities, since with increasing substrate concentration the specificenzyme activities do not approach v_(max) asymptotically, but decreaseagain. The maximum attainable enzyme activities, i.e. the specificenzyme activities at the maxima of the kinetic curves, are ˜13 U/mg forthe wild-type protein and ˜19 U/mg (G39A) and ˜20 U/mg (G39S) for themutant proteins. The enzyme activities that can be reached at thesubstrate concentrations used for whole-cell biotransformation are moreinteresting than the maximum attainable enzyme activities. As anexample, these are to be compared for the substrate concentration of 10mM dehydrocholic acid, and are ˜6.7 U/mg for the wild-type protein, ˜13U/mg for the G39A mutant and ˜18 U/mg for the G39S mutant. At 10 mMsubstrate concentration, the G39A mutant would accordingly be twice asactive as the wild-type protein, and the G39S mutant would even haveabout 3 times its activity. These differences should be even morepronounced at higher substrate concentrations, as the wild-type proteindisplays stronger substrate inhibition than the G39A mutant, and theG39A mutant is in its turn more strongly substrate-inhibited than theG39S mutant.

Production Example 4: Production and Characterization of the FDH D221GMutant

4.1 Cloning pET21a(+) FDH

4.1.1 PCR Amplification of Mycobacterium vaccae Formate Dehydrogenase

The template used for the amplification is genomic DNA of Mycobacteriumvaccae, which was obtained from the German Collection of Microorganismsand Cell Cultures (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, DMSZ), Brunswick. The primers for the amplificationwere

fdh_for  (SEQ ID NO: 23) (5′-CGATCATATGGCAAAGGTCCTGTGCGTTC-3′) andfdh_rev  (SEQ ID NO: 24) (5′-GCTAGAATTCTCAGCCGCCTTCTTGAACT-3′),obtained from Eurofins MWG GmbH, Ebersberg. The recognition sites forthe restriction enzymes are underlined. The rev-primer contains theEcoRI cleavage site and the for-primer contains the NdeI cleavage site.The PCR assays and PCR programs are shown in the following table.

TABLE PCR assay for the amplification of formate dehydrogenase fromMycobacterium vaccae Component Volume [μl] 10x Taq buffer (with Mg²⁺⁾ 5dNTPs (10 mM) 1 Fdh_for (100 μM) 0.5 Fdh_rev (100 μM) 0.5 Template DNA(≥100 ng/μL) 1 Taq DNA polymerase (5 U/mL) 0.5 Distilled water 41.5

TABLE PCR program for the amplification of formate dehydrogenase fromMycobacterium vaccae Seg- Cycle ment number Denaturation AnnealingElongation 1 1 94° C., 2 min 2 5 94° C., 30 s 55.6° C., 30 s 72° C., 75s 3 25 94° C., 30 s 58.6° C., 30 s 72° C., 75 s 4 1 72° C., 75 s4.1.2 Restriction Digestion of pET21a(+) and FDH PCR Product

5 μl 10× NEBuffer EcoRI, 2.5 μl NdeI (20 U/mL) and 2.5 μl EcoRI (20U/mL) (in each case New England Biolabs, Frankfurt) were added to 1-5 μgof DNA (pET21a(+) or FDH PCR product) dissolved in water, and made up to50 μl total volume with distilled water. The preparations were in eachcase incubated for 1 h at 37° C. Then the cut DNA fragments were appliedto 1% agarose gel (1% (w/v) agarose, 0.05% (v/v) ethidium bromide) andthe DNA fragments were separated electrophoretically for 55 minutes at120 V. Then the bands of the correct size (1.2 kb for the FDH-gene, 5.4kb for the pET21a(+)-plasmid) were cut out of the agarose gel with ascalpel and were isolated using the QIAquick Gel Extraction Kit (QIAGEN,Hilden) according to the manufacturer's protocol

4.1.3 Ligation of Cut pET21a(+) and FDH

1 μl T4 ligase (3 U/μL) and 1 μl 10× ligase buffer (in each case NewEngland Biolabs, Frankfurt) were added to 100 ng of cut vector DNA and111 ng of cut FDH DNA and made up to a total volume of 10 μl withdistilled water. The ligation preparation was incubated overnight at 4°C.

4.1.4 Transformation of the Ligation Preparation into ChemicallyCompetent E. coli DH5a

At the end of the ligation step, the 10 μL ligation preparation is addedto 200 μl of chemically competent E. coli DH5α prepared according to thestandard protocol. Next there is a 30-min incubation step on ice,followed by heat shock at 42° C. (90 seconds). Then 600 μl of sterile LBmedium is added to the transformation preparation and the cells areincubated at 200 rpm and 37° C. in a shaking incubator for 45 minutes.In the next step, the preparation is centrifuged at 3000 rpm for 60seconds in a benchtop centrifuge, 700 μl of the supernatant isdiscarded, the cells are resuspended in the remaining supernatant andplated out on an LB-agar plate with 100 mg/l ampicillin. The agar plateis then incubated overnight at 37° C.

4.2. Production of pET21a(+) FDH D221G

The production of an FDH mutant, which can regenerate not only NADH, butalso NADHP, will be explained in more detail with the mutant D221G.

Aspartate (D) 221 is an amino acid with a negatively-charged large sidechain, which is located directly next to the arginine (R) residue, bywhich NADP⁺ is to be bound. This can lead to repulsion of the phosphategroup in the NADP⁺, which is also negatively charged. The aspartate istherefore replaced with the small uncharged amino acid residue glycine(G).

4.2.1 Primers Used

mt1: (SEQ ID NO: 30) 5′-C CTG CAC TAC ACC G G C CGT CAC CGC CTG C-3′ NI_fdh_R: (SEQ ID NO: 31) 5′-GCTCGAATTCTCAGACCGCCTTC-3′ 4.2.2 Procedure

First a set of two complementary megaprimers was produced using themt-primer and the primer NI_fdh_R.

Plasmid pET21a(+)FDH was used as template. The PCR program used is shownin the following table:

TABLE Megaprimer PCR program Seg- Cycle ment number DenaturationAnnealing Elongation 1 1 94° C., 2 min 2 30 94° C., 30 s 60° C., 30 s72° C., 40 s 3 1 72° C., 5 min

By combining primer mt1 with the primer NI_fdh_R, the length of themegaprimer becomes 650 bp. With this PCR product of the first PCR, gelelectrophoresis and isolation of the desired band from the gel arecarried out. A second PCR is carried out as whole plasmid PCR with themegaprimers as primers and the plasmid DNA (pETfdh) as template. Thereaction mixture and the temperature scheme for the whole plasmid PCRare shown in the following tables. The 2× EZClone enzyme mix, theEZClone solution 1, the 1.1 kb marker and the DpnI were obtained fromthe GeneMorph II EZClone Domain Mutagenesis Kit (Stratagene).

TABLE Preparation for a MEGA WHOP PCR (total volume 50 μL) ComponentMegaprimer 250 ng (~2.5 μL from a standard-PCR) Template (pETfdh) 50 ng2x EZClone enzyme mix 25 μL EZClone solution 1 3 μL distilled water to50 μL

The first step in the PCR program (68° C., 5 min) is for removing thebases appended nonspecifically by the Taq-polymerase by the 3′→5′exonuclease activity of the polymerase used in the MEGA WHOP PCR.

TABLE MEGA WHOP PCR program Seg- Cycle ment number DenaturationAnnealing Elongation 1 1 68° C., 5 min 2 1 95° C., 1 min 3 25 95° C., 50s 60° C., 50 s 68° C., 13 min

The PCR product is a double-stranded plasmid with single-strand breaks,which are only closed in E. coli. 10 U DpnI were added to the 50 μL PCRproduct and the preparation was incubated at 37° C. for two hours. DpnIonly degrades methylated DNA, i.e. the template DNA used, but not themegaprimer or the synthesized plasmid. The template plasmid must beproduced with a dam⁺ strain (such as DH10B or JM109), to obtainmethylated starting DNA.

4.3 Expression and Isolation of the Mutant D221G

First an LB preculture was inoculated from frozen stored material orwith a colony from an agar plate and the preculture was incubatedovernight. Incubation was carried out at 37° C. and 250 rpm. The OD ofthe preculture was determined and the culture was inoculated to an OD of0.1. On reaching an OD of 0.5-1 (after about 2.5 h), it was induced withIPTG (final concentration 1 mM). The cells were harvested three hoursafter induction.

The cells were disrupted mechanically with glass beads. For this, 0.5 mLof glass beads were added to 0.5 mL of cell sample in a 1.5-mL reactionvessel and the reaction vessel was shaken for 3 min at maximum frequency(30 s⁻¹) in a Retsch vibratory mill. After cell maceration, the glassbeads were centrifuged off (2 min, 13000 rpm). Before and aftermaceration, the culture was put on ice, to minimize protein denaturationthrough heating of the sample in the vibratory mill. This macerationprotocol is optimized for the use of samples frozen at −20° C.

4.4 Characterization of the Mutant D221G

Summary of kinetic constants of FDH at pH 6, 7 and 8.

K_(M) K_(M) Specific (mM) (mM) activity sub- co- Enzyme pH SubstrateCofactor (U × mg⁻¹⁾ strate factor FDH 6 Sodium formate NAD⁺ 4.6 46.860.83 6 Sodium formate NADP⁺ 1 0.3 7 Sodium formate NAD⁺ 5.1 45.56 0.61 7Sodium formate NADP⁺ 1.2 0.51 8 Sodium formate NAD⁺ 4.7 59.26 0.52 8Sodium formate NADP⁺ 1 1.01The data prove that the mutant produced can utilize both NAD⁺ and NADP⁺as cofactor.

Production Example 5: Production of an E. coli 7α-HSDH Knockout Mutant(E. coli BL21 (DE3) hdhA⁻ KanR⁺)

The target is deletion of the disturbing 7α-HSDH activity in theexpression strain E. coli BL21 (DE3).

With the method described below, an antibiotic resistance gene isinserted in the target gene of 7α-HSDH, so that the target gene isswitched off.

5.1 Sequence Information for 7α-HSDH from E. coli BL21(DE3)

Amino acid sequence: (SEQ ID NO:26)

Nucleotide sequence (SEQ ID NO:25)

Accession: NC_012971 REGION: 1642470 . . . 1643237

5.2 Primers Used

The following primers were prepared for switching off the 7α-HSDH fromE. coli BL21(DE3):

Primer for the Retargeting of the LI.LtrB Intron:

467|468a-IBS (SEQ ID NO: 27)AAAAAAGCTTATAATTATCCTTATAGGACGTCATGGTGCGCCCAGATAG GGTG 467|468a-EBS1d(SEQ ID NO: 28) CAGATTGTACAAATGTGGTGATAACAGATAAGTCGTCATGTTTAACTTACCTTTCTTTGT 467|468a-EBS2 (SEQ ID NO: 29)TGAACGCAAGTTTCTAATTTCGGTTTCCTATCGATAGAGGAAAGTGTCT Insertion Location467|468a GCAGCTTTAGATGATGCATAGGAAGTCATG-intron-TTTATATTTTT ATTT5.3 Preparation of the Knockout Mutant

The knockout mutant was prepared using the kit TargeTron™ Gene KnockoutSystem from Sigma Aldrich according to the manufacturer's instructions.The QIAquick PCR Purification Kit from Qiagen was used for purifying thePCR product according to step B.6. of the TargeTron™ Gene KnockoutSystem.

Ligation of the HindIII/BsrGI-digested intron PCR product into thelinearized pACD4K-C vector was carried out as follows: the reaction wascarried out overnight at 16° C.

20 μl Preparation:

2 μl pACD4K-C linear vector (40 ng) 6 μl HindIII/BsrGI-digested intronPCR product 2 μl ATP (10 mM) 2 μl ligase buffer (10 x) (Fermentas) 2 μlT4-ligase (Fermentas) 6 μl H₂O

5 μl of ligation reaction solution was added to 200 μl ofchemically-competent cells of E. coli BL21 (DE3) and incubated on icefor 20 min. Further transformation was carried out as described by themanufacturer.

The transformation preparations were plated out on LB-agar plates,containing 33 μg/mL kanamycin. Kanamycin-resistant cells were picked andthese were in each case inoculated over several nights in 5 ml LBovernight cultures (in each case with 5 μl of a kanamycin solution (33mg/ml)). Finally a 200 ml LB culture (with 200 μl kanamycin solution (33mg/mL)) was inoculated with an overnight culture and was incubated for 5h at 37° C. and 180 rpm in a shaking incubator. Then the temperature wasraised to 42° C. for 1 hour. This culture was used for inoculating a 5mL LB overnight culture (in each case with 5 μl of a kanamycin solution(33 mg/mL)). After incubation overnight at 37° C. and 180 rpm, theculture was streaked on an LB-agar plate with 33 μg/mL kanamycin. Afterovernight incubation at 37° C., colonies were picked and streaked onLB-agar plate with 33 μg/mL kanamycin and 34 μg/mL chloramphenicol.

After overnight incubation at 37° C., chloramphenicol-sensitive mutantswere found. This is necessary in order to confirm the loss of theplasmid that is carried by the inducible knockout system and is nolonger required after successful knockout.

5.4. Detection of the Knockout

The 7α-HSDH gene was amplified by colony PCR with the primers

7alpha-ko-check_fwd  SEQ ID NO: 32 (5′-TTAATTGAGCTCCTGTACCCCACCACC-3′) and 7alpha-ko-check_rev  SEQ ID NO: 33(5′-GTGTTTAATTCTGACAACCTGAGACTCGAC-3′).The resultant fragment had a length of approx. 2.5-3 kb and wassequenced with the primer 7alpha-ko-check_fwd. The sequencing showedthat the DNA sequence of 7α-HSDH is interrupted by an insert from thepACD4K vector, resulting in knockout of 7α-HSDH (sequencing data notshown).

Reaction Example 1: Enzymatic Conversion of 7-Keto-LCA by 7β-HSDH

For verification of the biochemical function of 7β-HSDH, conversion of7-keto-LCA by 7β-HSDH was carried out. The 20 ml reaction mixturecontains 50 mM 7-keto-LCA (approx. 0.4 g), 5 U/ml 7β-HSDH and 0.05 mMNADP⁺. 4 U/ml ADH and 1% isopropanol were used for regeneration of NADPH(see Scheme 1). The reaction was carried out in a fume cupboard at pH8and 24° C. with stirring. As acetone evaporates more quickly thanisopropanol, the reaction is shifted toward formation of UDCA. Further1% isopropanol was added after 24 h, 48 h and 72 h. The product wasanalyzed by TLC (silica gel 60, Merck, solvent petroleum ether and ethylacetate 1:10, vol:vol). In TLC, the product was compared with authenticreferences 7-keto-LCA, UDCA and CDCA. The TLC analysis shows that UDCAwas formed from 7-keto-LCA by the 7β-HSDH. The enantiomer CDCA is notdetectable in TLC.

Reaction Example 2: Enzymatic Production of 3,12-Diketo-7β-CA from DHCAby 7β-HSDH

To verify the usability of 7β-HSDH for preparing 12-keto-UDCA from DHCA,conversion of DHCA by 7β-HSDH was carried out. The 50 ml of reactionmixture contain 50 mM DHCA (1 g), 5 U/ml 7β-HSDH and 0.05 mM NADP⁺. 4U/ml ADH and 1% isopropanol were used for regeneration of NADPH (seeScheme 2). The reaction was carried out in a fume cupboard at pH8 and24° C. with stirring. As acetone evaporates more quickly thanisopropanol, the reaction is shifted toward formation of3,12-diketo-7β-CA. In order to achieve complete conversion, further 1%isopropanol was added after 24 h, 48 h and 72 h. The intermediate3,12-diketo-7β-CA was analyzed by TLC. The educt DHCA was no longerdetectable in TLC (silica gel 60, Merck; solventchloroform:methanol:acetic acid 10:1:0.08 vol:vol:vol).

Scheme 2: Schematic representation of reduction of DHCA by 7β-HSDH. TheADH regenerates the cofactor NADPH.

Reaction Example 3: Enzymatic Conversion of 3,12-Diketo-7β-CA to12-Keto-UDCA

The intermediate 3,12-diketo-7β-CA (produced according to reactionexample 2) was transformed further by 3α-HSDH (SEQ ID NO:5 and 6) fromComamonas testosteroni (Mobus, E. and E. Maser, Molecular Cloning,overexpression, and characterization of steroid-inducible3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonastestosteroni. A novel member of the short-chain dehydrogenase/reductasesuperfamily. J Biol Chem, 1998. 273(47): p. 30888-96) to 12-keto-UDCA.This 3α-HSDH requires cofactor NADH, which was regenerated by the FDH(see FIG. 3). 4 U/ml 3α-HSDH, 1 U/ml FDH (NADH-dependent, Codexis), 200mM sodium formate and 0.05 mM NAD⁺ were added to the reaction mixture.After 40 h the product was acidified with 2 M HCl to pH2 and extractedwith 6×10 ml ethyl acetate. After evaporation, 1.07 g of product wasobtained. The product 12-keto-UDCA was analyzed and confirmed by TLC andNMR. 3alpha-HSDH was prepared as for the preparation of 7β-HSDH, butwith the plasmid pET22b+, and was used without further purification.

Reaction Example 4: Chemical Reaction of CA to DHCA

1320 L of glacial acetic acid is put in a 2000 L stirred vessel and 110kg (260 mol) of cholic acid (CA) is dissolved therein. 422 L of sodiumhypochloride solution (2.3 molar) is added to this solution at 20 to 40°C. and the reaction solution is then stirred for at least a further 1hour for completion of the reaction. Dehydrocholic acid (DHCA) isisolated by centrifugation at a yield of 100 kg (90%).

Reaction Example 5: Chemical Reaction of 12-Keto-UDCA to UDCA

105 g (0.258 mol) of 12-keto-UDCA is dissolved in 384 ml triethyleneglycol, 52.2 g (1.304 mol) sodium hydroxide and 75.95 ml (1.563 mol)hydrazine hydrate and heated slowly to 180° C. There is formation ofhydrazone, which starting from 160° C. is transformed with splitting offof nitrogen to UDCA. The reaction mixture is held at 180° C. for 8 hoursfor completing the reaction. The reaction mixture is cooled to below100° C., and 1500 ml water is added. Then the UDCA is precipitated byacidifying with hydrochloric acid. The product is obtained at a yield of96.2 g to 99.2 g (up to 95%-98%).

Reaction Example 6: Enzymatic Conversion of DHCA to 12-Keto-UDCA by7β-HSDH, FHD D221G and 3α-HSDH

The purpose of this example is to investigate whether a two-stepenzymatic conversion of DHCA to 12-keto-UDCA with simultaneous cofactorregeneration with an FDH mutant used according to the invention ispossible. As the FDH mutant D221G used accepts both NADP⁺ and NAD⁺ ascofactor, for the NADH-dependent 3α-HSDH it is not necessary for thereaction mixture to contain any additional cofactor regeneration system.

Examples of two partial reactions are illustrated graphically below.FDH⁺ designates the mutant FDH D221G.

For this, the enzymes 7β-HSDH from Collinsella aerofaciens, 3α-HSDH fromComamonas testosteroni and the FDH mutant D221G derived from FDH fromMycobacterium vaccae were expressed separately from one another in amodified E. coli expression strain and were used for the reaction. TheE. coli strain used for expression was modified so that it does notexpress any 7α-HSDH enzyme activity. This side activity, widelyoccurring in E. coli, can lead, in reactions of the present type(stereospecific conversion of the 7-keto group), to undesirable sidereactions and therefore to contamination of the reaction product to beproduced.

In the next experiment, the knock-out strain E. coli BL21(DE3)A7α-HSDHwas used, which is described in the applicant's earlier European patentapplication EP 10164003.5. Reference is expressly made hereby to thedisclosure of this patent application.

6.1 Plasmids Used

Plasmids for the expression of 7β-HSDH, 3α-HSDH and FHD D221G:

pET28a(+)-7β-HSDH,

pET22b(+)-3α-HSDH and

pET21a(+)-FDH-D221G.

6.2 Bacterial Strains and Culture Conditions:

The aforementioned knock-out strain E. coli BL21(DE3)A7α-HSDH wascultured at 37° C. in LB medium containing the necessary antibiotics.After induction with 0.5 mM IPTG on reaching OD₆₀₀=0.8, incubation wascontinued at 140 rpm for a period of 12 hours at 25° C.

6.3 Enzyme Overexpression and Purification

Overexpression of 7β-HSDH, 3α-HSDH and the FDH mutant D221G in E. coliBL21(DE3)A7α-HSDH and enzyme purification were carried out in the sameconditions as described above in production example 2 for theoverexpression and purification of 7β-HSDH in E. coli BL21 (DE3). Theyields per liter of culture medium (shaken flask at OD₆₀₀˜6) were asfollows:

7β-HSDH: 3883 U (for DHCA and NADPH)

3α-HSDH: 6853 U (for DHCA and NADH)

FDH mutant: 47 U (for sodium formate and NAD⁺).

Content and purity of the proteins were determined by SDS-PAGE anddensitometer scanning using Scion Image Beta 4.0.2 (Scion, USA).

6.4 Preparative-Scale Enzymatic Synthesis of 12-Keto-UDCA

800 ml of a reaction mixture containing 7β-HSDH (2.4 U×ml⁻¹), 3α-HSDH(2.4 U×ml⁻¹), FDH D221G (0.325 U×ml⁻¹), NADP⁺ (10 μM), NAD⁺ (10 μM),sodium formate (250 mM), DHCA (10 mM, 3.2 g) and potassium phosphatebuffer (50 mM, pH 6) was stirred at 24° C. All three enzymes that wereemployed in this experiment were used as cell raw extracts without anadditional purification step. After 12 hours the reaction was stopped byremoving the enzymes by ultrafiltration using a membrane with a poresize of 10 kDa (Millipore, USA). The product in the filtrate waspurified by acidifying with hydrochloric acid to pH 2 followed by paperfiltration. After drying the product at 60° C. overnight, 2.9 g of thedesired product was obtained.

The product was analyzed by HPLC and NMR.

Analysis Data (Partial):

¹H NMR (deuterated DMSO, 500 MHz) δ=3.92 (2H, m, H-3α and H-7β) and

¹H NMR (deuterated DMSO, 125 MHz) δ=69.38 (CH, 3-C); δ=69.09 (CH, 7-C);δ=213.86 (C, 12-C)

Yield: 90.6%

Purity: 99%

Reaction Example 7: Whole-Cell Biotransformation of DHCA to 12-Keto-UDCAby Coexpression of FDH D221G, 7β-HSDH and 3α-HSDH in the Two-PlasmidSystem

The aim was to investigate whether a two-step whole-cell reduction ofdehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid (12-keto-UDCA)(cf. scheme according to reaction example 6, above) is possible.

For this, the knockout strain E. coli BL21 (DE3) hdhA⁻ KanR⁺ pET21a(+)FDH 7β-HSDH pCOLA(mod) 3α-HSDH prepared above was used, in which, inaddition to the 7β-HSDH and the mutant FDH D221G, a 3α-HSDH fromComamonas testosteroni is expressed recombinantly. As the FDH mutantused accepts both NADP⁺ and NAD⁺ as cofactor, for the NADH-dependent3α-HSDH it was not necessary to insert any additional cofactorregeneration system into the biotransformation strain.

7.1 Strain Used:

Escherichia coli BL21 (DE3) hdhA⁻ KanR⁺

7.2 Molecular Biology Procedures Used

7.2.1 Polymerase Chain Reaction

The polymerase chain reaction (PCR) was used for cloning the 7β-HSDH.The plasmid pET22b(+) 7β-HSDH served as template for amplification ofthe 7β-HSDH.

PCR reactions were carried out in 500 μL PCR tubes with 20 μL reactionvolume. The reactions were carried out in a Thermocycler from thecompany Eppendorf. For amplification of 7β-HSDH, in each case 4 μLHF-buffer, 1 μL template DNA, 1 μL each of forward and reverse primer(10 μM), 0.4 μL of deoxynucleotide triphosphate solution (10 mM) and 0.2μL of Phusion DNA polymerase (2 U/μL) were added. The volume of thepreparation was adjusted to 20 μL with RNase-free water.

For the reaction, first a 2-min initial denaturation step was carriedout at 98° C. Then 34 cycles of denaturation (30 s at 98° C.), primerhybridization (2 min at 48° C.) and elongation (2 min at 72° C.) werecarried out. As a last step, final elongation of 10 min was carried outat 72° C. before stopping the polymerase chain reaction by cooling to 4°C.

7.2.2 Purification of DNA Fragments by Gel Extraction

For purification of DNA fragments, first they were separated by agarosegel electrophoresis. The corresponding bands were made visible with UVlight, identified based on their size and cut out of the gel with ascalpel. Extraction was carried out with the QIAquick Gel Extraction Kitaccording to the manufacturer's protocol. 30-50 μL H₂O was used forelution of the purified DNA.

7.2.3 Restriction with Endonucleases

The restriction reactions were carried out in a total volume of 20-50μL. For this, 10-20 U of the respective restriction enzymes was added tothe DNA to be cut. In addition, the reaction buffer recommended by themanufacturer was used and optionally 0.5 μL of bovine serum albumin(BSA, 10 mg/mL) was added, based on the manufacturer's recommendation.Restriction digestion was carried out for 2 h at 37° C. Then therestricted fragments were purified either by gel extraction (vectordigestion products) or using the QIAquick PCR Purification Kit (digestedPCR products).

7.2.4 Purification of DNA Fragments Using the QIAquick PCR PurificationKit

In order to purify restricted PCR fragments, DNA was purified using theQIAquick PCR Purification Kit. Purification was carried out according tothe manufacturer's instructions, using 30-50 μL H₂O for elution of thepurified DNA.

7.2.5 Ligation of DNA Fragments

For cloning restricted DNA fragments into expression vectors, both DNAmolecules were cut with the same restriction enzymes. By using twodifferent enzymes, on the one hand religation of the vector can beprevented, and on the other hand the insert can be incorporated in adefined orientation. The enzyme used was T4-DNA-ligase, which catalyzesthe formation of a phosphodiester bond between a free 5′-phosphate groupand a free 3′-OH end of a deoxyribonucleic acid.

For cloning the expression constructs, in each case 4 μL of agene-coding, purified DNA fragment with restricted ends was added to 12μL of a restricted, purified vector. Then 2 μL 10× ligase buffer, 1 μLadenosine triphosphate (ATP, 1 mM) and 1 μL T4-DNA-ligase (400 U/μL)were added. The reactions continued overnight at 16° C.

7.3 Vector Constructs Used

7.3.1 pET21a(+) FDH 7β-HSDH

For this vector construct, 7β-HSDH was cloned into pET21a(+) FDH, sothat the 7beta-HSDH coding gene is located downstream of the FDH. Forthis purpose, a stop codon had to be inserted in the FDH at the 5′-end.Compared with the original sequence (Lys-Lys-Ala-Val-stop), in thisconstruct the C-terminal valine residue was replaced with an alanineresidue and a further three amino acids were appended, resulting in thefollowing C-terminal sequence: Lys-Lys-Ala-Ala-Gly-Asn-Ser-stop.Moreover, for increased translation, an additional ribosomal bindingsite was inserted in 7β-HSDH between FDH and 7beta-HSDH.

The primers 7beta_fwd_EcoRI and S_7beta_rev_HindIII were used for PCRamplification of 7β-HSDH.

7beta_fwd_EcoRI (SEQ ID NO: 13): 5′-GC GAATTC G TGA AAGGAG ATATACATGAACCTGAGGGAGAAG TACGG-3′ S 7beta_revHindIII (SEQ ID NO: 3): 5′-CCCAAGCTT CTAGTCGCGGTAGAACGA-3′The hybridizing sequence regions (bold), restriction sites (boldunderlined), ribosomal binding sites (underlined), and fillingnucleotides (italics) are shown. In the primer 7beta_fwd_EcoRI,additionally a stop codon (double underlined) and a nucleotide forreading frame shift (bold) were added.Cloning was carried out via the EcoRI and HindIII cleavage sites.7.3.2 pCOLA(mod) 3α-HSDH

In order to be able to reduce dehydrocholic acid in two steps to12-keto-ursodeoxycholic acid, in addition to the cofactor regenerationsystem FDH, the enzymes 7β-HSDH and 3α-HSDH must also be present in acell. To make this possible, the vector construct pCOLA(mod) 3α-HSDH, amodified derivative of the pCOLA-duet vector, was prepared, which iscotransformable with pET-vectors.

For this purpose, the 3α-HSDH coding gene was cut out via the NdeI andBlpI cleavage sites from the vector pET22b(+) 3α-HSDH and, via the samecleavage sites, cloned into MCS2 of the pCOLA(mod) vector. Aftersequencing, it was found that at position 45 of the DNA sequence, aguanine was replaced with a cytosine, but this results in a silentmutation. However, this mutation has no effect on the amino acidsequence; therefore this is a so-called silent mutation.

Both vectors were cotransformed into the aforementioned strain, asdescribed below. The genes are IPTG-inducible.

7.4 Heat-Shock Transformation of E. coli Cells

For this purpose, 200 μL of chemically-competent E. coli BL21 (DE3)hdhA⁻ KanR⁺ or DH5α were thawed and 1 μL DNA was added. These were firstincubated on ice for 45 min. Then the cells were submitted to heat shockfor 45 s at 42° C. Then 600 μL of LB medium was added to the cells andthey were shaken at 37° C. and 200 rpm, so that they could develop thedesired antibiotic resistance. Next the cells were centrifuged in abenchtop centrifuge at 3000 rpm for 2 min, after which 150 μL of thesupernatant was discarded. The cells were resuspended in the remainingsupernatant and plated out on LB-agar plates with the correspondingantibiotic. Then the plates were incubated overnight at 37° C.

In the case of cotransformation of two different plasmids, as isnecessary for creating the strain that contains the plasmids pCOLA(mod)3α-HSDH and pET21a(+) FDH 7β-HSDH, in each case 2 μL of the plasmids tobe inserted was added to 50 μL of chemically-competent E. coli BL21(DE3) hdhA⁻ KanR⁺. After transformation, the cells were added topreculture tubes with 5 mL of LB medium with the correspondingantibiotic and were incubated at 37° C. on the preculture shaker for16-48 h at 200 rpm, until growth of bacteria in the preculture tube wasdetected. Next, 5 μL of the bacterial suspension was plated out onLB-agar plates with the corresponding antibiotic and was incubatedovernight at 37° C.

7.5 Cultivation of the Strain in the Shaken Flask:

For expression of recombinant proteins, the corresponding E. coli BL21(DE3) hdhA⁻ KanR⁺ comprising the two expression plasmids was incubatedovernight at 37° C. and 200 rpm in 5 mL of LB medium with addition ofthe corresponding antibiotic. Then 200 mL of TB medium with addition ofthe corresponding antibiotic was inoculated with this preculture andincubated at 37° C., 250 rpm. On reaching OD600 of 0.6-0.8, expressionof the recombinant protein was induced with 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG) and the culture was incubatedat 25° C., 160 rpm for a further 21 h.

7.6 Carrying Out Whole-Cell Biotransformation and Test Result

The reactions were carried out in suspension in 2 mL reaction volume atpH 6.0, using a cell density of OD₆₀₀=20. 25 mM or 50 mM dehydrocholicacid was selected as the substrate concentration, and the cosubstrateconcentration used was 400 mM. The tests were carried out at 25° C. withexclusion of air. Samples were taken after 48 h.

In the case of the reaction mixture with 25 mM, substrate is no longerdetectable, whereas for the reaction mixture with 50 mM dehydrocholicacid, a substrate peak is detectable, but its peak area is below thelimit of 1 μg/mL substrate in the sample for which the HPLC method iscalibrated. The same applies to the peaks of 3,12-diketo-ursodeoxycholicacid. The largest peak in the chromatograms is that of the product12-keto-ursodeoxycholic acid.

Thus, it can be shown, surprisingly, that a two-step whole-cellreduction of dehydrocholic acid to 3-keto-ursodeoxycholic acid ispossible.

The chromatograms of the HPLC measurement are shown in FIG. 7.

The HPLC analysis was carried out as follows: The chromatography columnused was the reverse-phase chromatography column Hi-bar Purospher 125-4RP-18e (5 μm) from the company Merck, Darmstadt. In this case a nonpolarphase serves as stationary phase, whereas a polar phase forms the mobilephase. The method of gradient elution was used for the HPLC analysis. Anincreasing proportion of acetonitrile is added to the eluent, phosphoricacid water, (pH 2.6), and the acidification of the solvent causes auniform degree of protonation of the analytes to be investigated. Afterelution of all components, the chromatography column is equilibratedwith the starting ratio of the two solvents. Gradient elution wascarried out by first pumping a solvent mixture with a constantcomposition of 65% (v/v) of phosphoric acid water and 35% (v/v) ofacetonitrile through the HPLC column for 3 min. From minute 3 to 7.5,the proportion of acetonitrile is increased linearly to 39% (v/v).Between minute 7.5 and 10 there is another linear increase of theproportion of acetonitrile to 40% (v/v). For elution of all samplecomponents, the proportion of acetonitrile is increased between minute10 and 11 also linearly to 70% (v/v) and held constantly at this valuefor a further two minutes. After that, the proportion of acetonitrile isreduced from minute 13 to 15 back to the initial value of 35% and thecolumn is equilibrated with this solvent composition for 3 min. The flowis set at 1.000 mL/min for the total duration. Eluted analytes aredetected by a UV-detector at a wavelength of 200 nm.

Reaction Example 8: Whole-Cell Biotransformation of DHCA to 12-Keto-UDCAby Coexpression of FDH D221G, 7β-HSDH and 3α-HSDH in the Single-PlasmidSystem

The aim was to investigate whether a two-step whole-cell reduction ofdehydrocholic acid to 12-keto-ursodeoxycholic acid in a cellularsingle-plasmid system is possible.

8.1 Plasmids Used:

SEQ Designation Sequence ID NO 3alpha_fwd_HindIII 5′-CCCAAGCTTAAGGAGATATACATGTCCATCATCGTGATAAGCG-3′ 16 3alpha_rev_NotI5′-ATAAGAATGCGGCCGCTCAGAACTGTGTCGG GCG-3′ 17 7beta_mut_G39A_fwd5′-CGTCGTCATGGTCGCCCGTCGCGAGG-3′  9 7beta_mut_G39A_rev5′-CCTCGCGACGGGCGACCATGACGACG-3′ 10Restriction sites are underlined; ribosomal binding sites are bold.8.2 Production of the Vector:

The plasmid construct pET21a(+) FDH 7β-HSDH(G39A) 3α-HSDH (FIG. 8) isprepared as follows: Starting from the plasmid pET21a(+) FDH 7β-HSDH thewild-type 3α-HSDH (C. testosteroni; SEQ ID NO: 5, 6) was cloned in after7β-HSDH via the HindIII and NotI cleavage sites. For amplification of3α-HSDH, the primers 3alpha_fwd_HindIII and 3alpha_rev_NotI were used,with the plasmid pET22b(+) 3α-HSDH serving as template for this. Thenthe G39A mutation was inserted into 7β-HSDH by site-directed mutagenesisaccording to the QuikChange protocol. The mutagenesis primers7beta_mut_G39A_fwd and 7beta_mut_G39A_rev were used.

8.3 Reaction:

The plasmid prepared was transformed into E. coli BL21 (DE3) hdhA⁻KanR⁺. With the resultant strain E. coli BL21 (DE3) hdhA⁻ KanR⁺pET21a(+) FDH 7β-HSDH(G39A) 3α-HSDH, whole-cell reactions were carriedout under the following conditions at the 150-mL scale: cell density OD30, 50 mM DHCA, 750 mM sodium formate, suspended in 50 mM potassiumphosphate buffer (pH 6.5) as cell and substrate suspension. Thereactions were carried out for 3.5 h at 25° C. The results of the HPLCanalysis (for procedure see reaction example 7, above) are shown in FIG.9.

Production Example 6: Production of Further 7β-HSDS Mutants andCharacterization Thereof

One possibility for generating an NADH-specific 7β-HSDH consists ofmodifying the available enzyme by various techniques of mutagenesis.Therefore, using site-directed mutagenesis, individual amino acids of7β-HSDH were to be substituted with others, which cause change of thecofactor specificity of 7β-HSDH from NADPH to NADH, so thatadvantageously NADP can be replaced with the less expensive NAD.

6.1 Primers

Altogether, the 7β-HSDH mutants G39D, G39D/R40L, G39D/R40I and G39D/R40Vwere produced. The G39D mutant is produced by Quickchange mutagenesis,and the other mutants are produced by the PCR method of Sanchis et al.(Sanchis J, Fernández L, Carballeira J D, Drone J, Gumulya Y, HöbenreichH, Kahakeaw D, Kille S, Lohmer R, Peyralans J J, Podtetenieff J, PrasadS, Soni P, Taglieber A, Wu S, Zilly F E, Reetz M T. Improved PCR methodfor the creation of saturation mutagenesis libraries in directedevolution: application to difficult-to-amplify templates. Appl MicrobiolBiotechnol. 2008 November; 81(2): 387-97). A sequence comparison of thewild type and of the mutants produced is shown in FIG. 10. The followingprimers were used for the mutagenesis, with the bases shown in italicscoding in each case for the exchanged amino acid:

G39D 7beta mut G39D fwd (SEQ ID NO: 41): CGTCGTCATGGTCGACCGTCGCGAGG7beta mut G39D rev (SEQ ID NO: 42):  CCTCGCGACGGTCGACCATGACGACGG39D R40L 7beta mut G39D R40L fwd (SEQ ID NO: 43): CGTCGTCATGGTCGACCTGCGCGAGG 3alphamut_AntiMid_rev (SEQ ID NO: 44): CCGCCGCATCCATACCGCCAGTTGTTTACCC G39D R40I7beta mut G39D R40I fwd (SEQ ID NO: 45): CGTCGTCATGGTCGACATTCGCGAGG3alphamut_AntiMid_rev (SEQ ID NO: 44):  CCGCCGCATCCATACCGCCAGTTGTTTACCCG39D R40V 7beta mut G39D R40V fwd (SEQ ID NO: 46): CGTCGTCATGGTCGACGTTCGCGAGG 3alphamut_AntiMid_rev (SEQ ID NO: 44): CCGCCGCATCCATACCGCCAGTTGTTTACCC6.2 Enzyme-Kinetic Investigation of the 7β-HSDH Mutants

The mutants prepared are evaluated by means of enzyme-kineticinvestigations, the results of which are shown as graphs in FIG. 11. 0.1mM NADPH is used as cofactor for investigating the unmodified 7β-HSDH,but 0.5 mM NADH is used for investigating the 7β-HSDH mutants. The needto increase the cofactor concentration in the enzyme-kineticinvestigation of the 7β-HSDH mutants is due to the increasedsemisaturation concentrations for the cofactor NADH in the mutants. Fromthe plots of substrate concentration versus specific enzyme activity,the characteristic curves of Michaelis-Menten kinetics can be seen forthe 7β-HSDH mutants, whereas the curve of Michaelis-Menten kinetics withsubstrate inhibition can be seen from the plot for the unmodifiedwild-type 7β-HSDH. Accordingly, the classical Michaelis-Menten model(equation 1) was used for evaluating the measurement series of the7β-HSDH mutants and the Michaelis-Menten model with substrate inhibitionwas used for the measurement series of the unmodified wild-type 7β-HSDH(equation 2).

$\begin{matrix}{{EA}_{x} = {v_{\max} \cdot \frac{c_{S}}{K_{m} + c_{S}}}} & {{Equation}\mspace{14mu} 1\mspace{14mu}\left( {{Michaelis}\text{-}{Menten}\mspace{14mu}{equation}} \right)}\end{matrix}$EA_(x): specific enzyme activity, U mg⁻¹=μmol min⁻¹ mg⁻¹v_(max): maximum specific enzyme activity, U mg⁻¹=μmol min⁻¹ mg⁻¹c_(s): substrate or cofactor concentration, mol L⁻¹K_(m): semisaturation concentration, mol L⁻¹

 Equation  2  (Michaelis-Menten  equation  with  substrate  inhibition)${EA}_{x} = {v_{\max} \cdot \frac{c_{S}}{K_{m} + {\left( {1 + \frac{\left( c_{S} \right)}{\left( K_{i} \right)}} \right)c_{S}}}}$EA_(x): specific enzyme activity, U mg⁻¹=μmol min⁻¹ mg⁻¹v_(max): maximum specific enzyme activity, U mg⁻¹=μmol min⁻¹ mg⁻¹c_(s): substrate or cofactor concentration, mol L⁻¹K_(m): semisaturation concentration, mol L⁻¹K: inhibition constant, mol L⁻¹The following table gives enzyme-kinetic parameters of the unmodifiedwild-type 7β-HSDH and the mutants thereof with altered cofactorspecificity. NADPH is used as cofactor for the wild type, whereas NADHis used as cofactor for the mutants.

K_(m), DHCA, K_(i), DHCA, v_(max), μM μM U mg−1 Wild type 31 ± 5  8600 ±1500 14.6 ± 1.0  G39D 660 ± 120 n.d. 2.90 ± 0.16 G39D R40I 920 ± 170n.d. 4.64 ± 0.27 G39D R40V 880 ± 120 n.d. 2.69 ± 0.12 G39D R40L 560 ±80  n.d. 1.60 ± 0.07

Production Example 7: Production of Further 7β-HSDS Mutants andCharacterization Thereof

Further 7β-HSDS mutants were produced in a preparation parallel toproduction example 6.

7.1 Primers

The mutagenesis primers shown below were used for the site-directedmutagenesis of 7β-HSDH. The primers were selected on the basis of the7β-HSDH gene sequence, so that they bring about the desired amino acidexchange. It was noted that the base to be mutated is localizedcentrally in the primer, and that the melting points of the primer pairsare located in the same region.

The following primer pairs were used for preparing the mutants:

TABLE Primers used for the site-directed mutagenesis of 7β-HSDHDesignation Position Primer 5′→3′ Sequence G39D_for G39D forwardGTCGTCATGGTCGACCGTCGCGAGGAG G39D_rev reverse CTCCTCGCGACGGTCGACCATGACGACG39D/RT40I_for G39D/R40I forward GTCATGGTCGACATTCGCGAGGAG G39D/R40I_revreverse CTCCTCGCGAATGTCGACCATGAC R40D_for R40I forwardGTCATGGTCGGCGATCGCGAGGAGAAG R40D_rev reverse CTTCTCCTCGCGATCGCCGACCATGACR40D/R41I_for R40D/R41I forward GTCATGGTCGGCGATATCGAGGAGAAGCTGR40D/R41I_rev reverse CAGCTTCTCCTCGATATCGCCGACCATGAC DIN_forG39D/R40I/R41N forward ATGGTCGACATTAACGAGGAGAAGCTG DIN_rev reverseCAGCTTCTCCTCGTTAATGTCGACCAT7.2 PCR Program

In the reaction, first there was a 2-min initial denaturation step at98° C. This was followed by 23 cycles of denaturation (30 s at 98° C.),primer hybridization (1 min at 60-68° C.) and elongation (3.5 min at 72°C.). As the last step, a final elongation was carried out for 10 min at72° C. before the polymerase chain reaction was stopped by cooling to 4°C.

7.3 PCR Assay

TABLE PCR assay for the production of the different 7β-HSDH variants HFbuffer (5x)  10 μl dNTP mix (10 mM) 1.0 μl Forward primer (10 pmol/μl)  5 μl Reverse primer (10 pmol/μl)   5 μl Template 1.5 μl Phusionpolymerase 0.5 μl DMSO 2.5 μl ddH₂O 24.5 μl   50 μl A pET21a vector withthe 7β-HSDH (wild type) was used as template.7.4 Procedure

For targeted exchange of amino acids in protein sequences, the DNAsequence of the corresponding gene is submitted to site-directedmutation. For this, mutually complementary primers are used, which carrythe desired mutation in their sequence. N6-adenine-methylated,double-stranded plasmid DNA, which carries the gene to be mutated,serves as template. N6-adenine-methylated plasmid DNA is isolated fromdam E. coli strain, for example E. coli DH5.

The polymerase chain reaction is carried out as described above. Theprimers are lengthened complementarily to the template, so that plasmidswith the desired mutation are formed, which have a strand break. Incontrast to other PCR reactions, the increase in DNA yield is in thiscase only linear, as newly formed DNA molecules cannot serve as templatefor the PCR reaction.

On completion of the PCR reaction, the PCR product was purified using aPCR-Purification-Kit (Analytik Jena) and the parental,N6-adenine-methylated DNA was digested with the restriction enzyme DpnI.This enzyme has the particular characteristic that it restrictsN6-adenine-methylated DNA nonspecifically, but not the newly formed,nonmethylated DNA.

Restriction was carried out by adding 1 μL DpnI to the PCR reactionmixture and incubating for 2 h or overnight at 37° C.

5 μl of this preparation were used for the transformation of 100 μl ofchemically-competent DH5α cells. After plasmid isolation, successfulmutation was confirmed by sequencing.

7.5 Characterization

The mutation was introduced into 7β-HSDH by site-directed mutagenesisaccording to the QuikChange protocol. The mutagenesis primers from thetable shown in Section 7.1 were used.

Using the assays given in the “Methods” section for photometricmeasurement of activity, the activity of the mutated enzymes wasmeasured in the presence of NADPH or NADH. The activities found arepresented in the following table.

TABLE Activity found for the different 7β-HSDH variants Volumetricactivity Specific activity [U/ml] [U/mg] Mutants produced NADPH NADHNADPH NADH G39D 119 2 5.1 0.1 G39D/R40I 0 21 0 0.8 R40D 0 3 0 0.1R40D/R41I 0 3 0 0.2 G39D/R40I/R41N 0 6 0 0.3 7β-HSDH (WT) 134 0 5.6 0

Reaction Example 9: Using NADH-Dependent 7β-HSDH in the Whole-CellReduction of Dehydrocholic Acid to 3,12-Diketo-UDCA

The use of NADH-dependent 7β-HSDH, produced in production example 6, inthe whole-cell biocatalytic conversion of DHCA to 3,12-diketo-UDCA isdemonstrated below. To improve comprehension, a review of possiblereaction pathways and reaction products is shown in FIG. 12.

In the present case, the 7β-HSDH mutants together with an NADH-dependentformate dehydrogenase from Mycobacterium vaccae were inserted in anexpression vector. The vector into which 7β-HSDH (G39D) is insertedbears the designation pFr7(D), corresponding to SEQ ID NO:49; the vectorinto which 7β-HSDH (G39D R40L) is inserted bears the designationpFr7(DL), corresponding to SEQ ID NO:50; the vector into which 7β-HSDH(G39D R40I) is inserted bears the designation pFr7(DI), corresponding toSEQ ID NO:51, and the vector into which 7β-HSDH (G39D R40V) is insertedbears the designation pFr7(DV), corresponding to SEQ ID NO:52. A generalplasmid map of these vectors is shown in FIG. 13.

These vectors were transformed into the strain E. coli BL49 (identicalto E. coli BL21(DE3) hdhA⁻ KanR⁺). From these strains, firstly 5 mLovernight cultures were grown in LB medium with 100 mg L⁻¹ ampicillin.In each case 1 mL of these overnight cultures was transferred on thenext day into 200 mL of TB medium in shaken flasks with 100 mg L⁻¹ampicillin. These cultures were cultured at 37° C. and 250 rpm in theshaking incubator, until an OD of 0.6-0.8 was reached. Then inductionwas carried out with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG).The subsequent expression phase was carried out for 21 h at 25° C. and160 rpm. The cells were harvested by 10-minute centrifugation at 3220 g.

Whole-cell biotransformation reactions were set up with the cellsproduced in this way. The concentrations of the substrate DHCA and ofthe product 3,12-diketo-UDCA in the reaction mixture at various timepoints was determined by high-performance liquid chromatography (HPLC).

The reaction mixtures for the biotransformations contained 17.7 g L⁻¹_(BTM) whole-cell biocatalysts, 100 mM substrate (DHCA), 500 mM sodiumformate and are suspended in 50 mM potassium formate buffer (pH 6.5).The process took place at the 20 mL scale as a batch process without pHmonitoring. The concentrations of product and substrate after 24 h areshown in FIG. 14. It can be seen that all mutants are suitable for thewhole-cell biocatalytic conversion of DHCA to 3,12-diketo-UDCA.

Reaction Example 10: Using NADH-Dependent 7β-HSDH in the Two-StepWhole-Cell Reduction of Dehydrocholic Acid to 12-Keto-UDCA

The use of NADH-dependent 7β-HSDH in the two-step whole-cellbiocatalytic conversion of DHCA to 12-diketo-UDCA is to be demonstratedbelow. The mutants 7β-HSDH (G39D) together with an NADH-dependentformate dehydrogenase from Mycobacterium vaccae and an NADH-dependent3α-HSDH from Comamonas testosteroni were inserted into an expressionvector. The use of 7β-HSDH (G39D) is shown as an example for allNADH-dependent 7β-HSDHs. The vector bears the designation pFr3T7(D) andis shown schematically in FIG. 15.

These vectors were transformed into the strain E. coli BL49 (identicalto E. coli BL21(DE3) hdhA⁻ KanR⁻); the resultant strain bears thedesignation E. coli BL49 pFr3T7(D). This strain was cultured in the 7 Lbioreactor at a working volume of 4 L in the defined minimal medium. Abrief initial phase at 37° C. was followed by a substrate-limitedexponential growth phase at 30° C. On reaching an optical density of25-30, protein expression of the cells was induced by adding 0.5 mMIPTG. Expression was then carried out for 24 h at 20° C. The cells (32.0g L⁻¹ _(BTM)) were harvested by centrifugation, resuspended in potassiumphosphate buffer (pH 6.5) and stored according to the standard protocolat −20° C.

Biotransformation was set up at the 20-mL scale in the followingreaction conditions: 100 mM DHCA, 17.7 g L⁻¹ _(BTM) of the storedbiocatalyst, 500 mM ammonium formate, 26% glycerol, 50 mM MgCl₂,suspended in 50 mM KPi buffer (pH 6.5). Using manual pH adjustment, thepH was maintained during the first 5 hours of the biotransformationprocess at pH 6.5. The bile salt concentrations are shown as a functionof time in FIG. 16. After 24 h, 92% of the product 12-keto-UDCA hadformed. The conversion of DHCA to 3,12-diketo-UDCA and the conversion of7,12-diketo-UDCA to 12-keto-UDCA show that all the reactions of 7β-HSDHshown in FIG. 12 are in fact catalyzed by 7β-HSDH (G39D).

Reaction Example 11: Two-Step Reduction of Dehydrocholic Acid in aSingle-Cell System

One route for the synthesis of ursodesoxycholic acid comprises thechemical oxidation of cholic acid to dehydrocholic acid with subsequentasymmetric enzymatic reductions of the 3-carbonyl group to the3α-hydroxyl group and of the 7-carbonyl group to the 7β-hydroxyl groupfollowed by chemical removal of the 12-carbonyl group.

For this synthesis route, the enzymes 7β-hydroxysteroid dehydrogenase(7β-HSDH) and 3α-hydroxysteroid dehydrogenase (3α-HSDH) are required forcatalysis of the enzymatic reduction reactions. In the reaction, inaddition cofactors (NADH or NADPH) are consumed stoichiometrically, andin an economic process these must be regenerated. Enzymatic regenerationpossibilities are often preferred for this. Suitable enzymes includeformate dehydrogenases (FDH), glucose dehydrogenases (GDH),glucose-6-phosphate dehydrogenases (G6PDH), alcohol dehydrogenases (ADH)or phosphite dehydrogenases (PtDH).

In contrast to the use of isolated enzymes, in whole-cell biocatalysis,with the necessary enzymes within a host organism, typically aunicellular microorganism, it is not possible to add individual enzymesin individual amounts to the reaction medium. In this case the enzymeactivities must be balanced by modifying the expression level of allparticipating enzymes either by cultivation methods or by geneticmodification of the whole-cell biocatalyst.

In the present example, vectors were used according to the invention,which contain all three genes for 7β-HSDH, 3α-HSDH and FDH. The vectorsare constructed so that the expression level of the three genes in thedesignated host strain, especially whole-cell biocatalyst strains basedon Escherichia coli BL21(DE3), are adapted in such a way that all threeenzymes have enzyme activities that are as similar as possible.

The three genes required in the whole-cell reduction of dehydrocholicacid to 12-keto-ursodeoxycholic acid are located on one and the samevector. These are genes that code for the following enzymes: anNADPH-dependent 7β-HSDH from Collinsella aerofaciens, an NADH-dependent3α-HSDH from Comamonas testosteroni, a mutated FDH from Mycobacteriumvaccae that is both NAD- and NADP-dependent. The expression levels ofthese three genes are balanced by the genetic construct, so that optimalwhole-cell biocatalysis can be carried out. In the present example the7β-HSDH mutants G39A and G39S were inserted instead of an unmodifiedwild-type enzyme into a vector for the whole-cell biocatalysis. Thevectors bear the designations pF(G)r7(A)r3 and pF(G)r7(S)r3 and areshown in FIGS. 17a and b . FIG. 18 shows a schematic representation ofthe possible reaction pathways and reaction products.

These vectors according to the invention were transformed intoEscherichia coli production strains. These strains represent thewhole-cell biocatalyst. The host strain used is a modified E. coliBL21(DE3) with the designation E. coli BL49. However, the host organismis not restricted to this host strain. The E. coli BL49 transformed withpF(G)r7(A)r3 bears the designation E. coli BL49 pF(G)r7(A)r3, and the E.coli BL49 transformed with pF(G)r7(S)r3 bears the designation E. coliBL49 pF(G)r7(S)r3.

11.1 Two-Step Whole-Cell Reduction of Dehydrocholic Acid at the 20-mLScale

The biocatalysts were compared by whole-cell biotransformation at the20-mL scale. For this, first a 5 mL overnight culture was grown in LBmedium with 100 mg L-1 ampicillin. On the next day, 1 mL of thisovernight culture was transferred into 200 mL of TB medium in a shakenflask with 100 mg of L-1 ampicillin. These cultures were cultured at 37°C. and 250 rpm in the shaking incubator, until an OD of 0.6-0.8 wasreached. Then induction was carried out with 1 mMisopropyl-β-D-thiogalactopyranoside (IPTG). The subsequent expressionphase was carried out for 21 h at 25° C. and 160 rpm. The cells wereharvested by 10-minute centrifugation at 3220 g.

Whole-cell biotransformation reactions were set up with the cellsproduced in this way. The amount of the product (12-keto-UDCA) formed inthe reaction mixture was decisive for assessment of the cells. Theconcentrations of the substrate DHCA, of the intermediates3,12-diketo-UDCA and 7,12-diketo-UDCA and of the product 12-keto-UDCA inthe reaction mixture were determined by high-performance liquidchromatography (HPLC).

The reaction mixtures for the biotransformations contained 11.8 g L⁻¹_(BTM) whole-cell biocatalysts, 100 mM substrate (DHCA), 500 mM sodiumformate and were suspended in 50 mM potassium formate buffer (pH 6.5).The process was carried out at the 20 mL scale as a batch processwithout pH monitoring. The following table gives the results of thewhole-cell biotransformation described above after a process time of 5h. The two new strains E. coli BL49 pF(G)r7(S)r3 and E. coli BL49pF(G)r7(A)r3 are compared with a reference strain E. coli BL49 pF(G)r7pC3. With the reference strain E. coli BL49 pF(G)r7 pC3 only 5.7±1.0% ofproduct (12-keto-UDCA) was formed, whereas with the new biocatalysts38.4±8.2% 12-keto-UDCA was formed with the strain E. coli BL49pF(G)r7(S)r3 and 47.7±2.1% 12-keto-UDCA with the strain E. coli BL49pF(G)r7(A)r3. Therefore the product concentration in the reactionmixture had increased relative to the reference strain by a factor of6.7 (E. coli BL49 pF(G)r7(S)r3) and by a factor of 8.4 (E. coli BL49pF(G)r7(A)r3).

Accordingly, the following table shows proportions of bile salts inbiotransformation batches after 5 h when using 100 mM substrate (DHCA).The batches with the two new biocatalyst strains E. coli BL49pF(G)r7(A)r3 and E. coli BL49 pF(G)r7(S)r3 are shown in comparison withthe strain E. coli BL49 pF(G)r7 pC3 according to the prior art:

Proportions of bile salts, % 12-keto- 3,12-diketo- 7,12-diketo- UDCAUDCA UDCA DHCA E. coli BL49  5.7 ± 1.0% 9.2 ± 0.2% 40.5 ± 1.9% 44.6 ±3.1%  pF(G)r7 pC3 E. coli BL49 47.7 ± 2.1% 2.4 ± 0.5% 48.9 ± 1.6% 1.0 ±0.0% pF(G)r7(A)r3 E. coli BL49 38.4 ± 8.2% 2.7 ± 2.7% 57.4 ± 4.4% 1.5 ±1.8% pF(G)r7(S)r311.2 Two-Step Whole-Cell Reduction of Dehydrocholic Acid at the 1 LScale

Through process control, product formation with the biocatalyst E. coliBL49 pF(G)r7(A)r3 was increased relative to the milliliter scale. Forthis, the biocatalyst was cultured in the 7 L bioreactor at a workingvolume of 4 L in the defined minimal medium. After a brief initial phaseat 37° C., there was a substrate-limited exponential growth phase at 30°C. On reaching an optical density of 25-30, protein expression of thecells was induced by adding 0.5 mM IPTG. Expression was then carried outfor 10 h at 25° C., before the cells were harvested by centrifugationand were stored at −20° C.

The biotransformation was set up in a 1 L bioreactor in the followingreaction conditions: 70 mM DHCA, 17.7 g L⁻¹ _(BTM) of the storedbiocatalyst, 500 mM sodium formate, 26% (v/v) glycerol, 50 mM MgCl₂,suspended in 50 mM KPi buffer (pH 6.5). Using pH adjustment, the pH wasmaintained at pH 6.5 throughout the biotransformation. The variation ofthe bile salt concentrations as a function of time is shown in FIG. 19.After 21 h, 99.4% of product (12-keto-UDCA) had formed and only 0.6% ofthe by-product 3,12-diketo-UDCA was detectable.

Reaction Example 12: Two-Step Reduction of Dehydrocholic Acid in aSingle-Cell System Using a Glucose Dehydrogenase for CofactorRegeneration

In this example, in addition to an NADPH-dependent 7β-HSDH fromCollinsella aerofaciens (mutant G39A) and an NADH-dependent 3α-HSDH fromComamonas testosteroni, a GDH from Bacillus subtilis that is both NAD-and NADP-dependent for the cofactor regeneration is expressed in awhole-cell biocatalyst (single-cell system) for the two-step reductionof DHCA to 12-keto-UDCA. The nucleic acid sequence and the associatedamino acid sequence of this GDH are given as SEQ ID NO:47 and SEQ IDNO:48 respectively.

The vectors used for this bear the designations p3T7(A)rG and p7(A)T3rGand are shown in FIG. 20 and FIG. 21 respectively.

The vectors according to the invention were transformed into Escherichiacoli production strains. These strains represent the whole-cellbiocatalyst. The host strain used is a modified E. coli BL21(DE3) withthe designation E. coli BL49 (identical to E. coli BL21(DE3) hdhA⁻KanR⁺). However, the host organism is not restricted to this hoststrain. The E. coli BL49 transformed with p7(A)T3rG bears thedesignation E. coli BL49 p7(A)T3rG, and the E. coli BL49 transformedwith p3T7(A)rG bears the designation E. coli BL49 p3T7(A)rG.

With the newly produced biocatalysts, using a total of 17.7 g/L BTMbiocatalysts, 100 mM DHCA can be converted to an extent of 98% to12-keto-UDCA.

12.1 Two-Step Whole-Cell Reduction of Dehydrocholic Acid at the 20-mLScale

The biocatalysts were compared by whole-cell biotransformation at the20-mL scale. Cultivation and determination of the concentration of thesubstrate DHCA, of the intermediates 3,12-diketo-UDCA and7,12-diketo-UDCA and of the product 12-keto-UDCA in the reaction mixturewere carried out as described in reaction example 11.1.

The reaction mixtures for the biotransformations contained 11.8 g/L BTMwhole-cell biocatalysts, 100 mM substrate (DHCA), 500 mM glucose andwere suspended in 50 mM potassium formate buffer (pH 7.3). The processtook place at the 20 mL scale as a batch process without pH monitoring.Perforation of the biocatalysts was not necessary. The following tableshows the results of the whole-cell biotransformation described aboveafter a process time of 24 h, showing the results of the whole-cellbiotransformation with the two strains E. coli BL49 p7(A)T3rG and E.coli BL49 p3T7(A)rG. With the new biocatalysts, using 11.8 g L⁻¹ _(BTM)biocatalyst, within 24 h 100 mM dehydrocholic acid (DHCA) can beconverted to an extent of 46% (E. coli BL49 p3T7(A)rG) or 68% (E. coliBL49 p7(A)T3rG) to 12-keto-ursodeoxycholic acid (12-keto-UDCA). Thefollowing table shows proportions of bile salts in biotransformationbatches after 24 h, using 100 mM substrate (DHCA) (batches with the twonew biocatalyst strains E. coli BL49 p3T7(A)rG and E. coli BL49p7(A)T3rG):

Proportions of bile salts, % 12-keto- 3,12-diketo- 7,12-diketo- UDCAUDCA UDCA DHCA E. coli BL49 46 ± 6%   7 ± 1% 7 ± 1% 40.0 ± 2% p3T7(A)rGE. coli BL49 68 ± 19% 15 ± 3% 1 ± 1%   12 ± 10% p7(A)T3rG12.2 Two-Step Whole-Cell Reduction of Dehydrocholic Acid with Cells ofthe Strain E. coli BL49 p7(A)T3rG Cultured in the Bioreactor at the20-mL Scale

The strain E. coli BL49 p7(A)T3rG was cultured in the 7 L bioreactor ata working volume of 4 L in the defined minimal medium. After a briefinitial phase at 37° C. there was a substrate-limited exponential growthphase at 30° C. On reaching an optical density of 25-30, proteinexpression of the cells was induced by adding 0.5 mM IPTG. Expressionwas then carried out for 24 h at 20° C. The cells (46.7 g/L BTM) wereharvested by centrifugation, resuspended in potassium phosphate buffer(pH 6.5) and stored according to the standard protocol at −20° C. At thetime of harvesting, the enzyme activities were: 16.4 U/mL 7β-HSDH, 3.6U/mL 3α-HSDH, 44.9 U/mL GDH (NADP), 18.1 U/mL GDH (NAD).

Biotransformation was set up at the 20-mL scale in the followingreaction conditions: 100 mM DHCA, 17.7 g/L BTM of the storedbiocatalyst, 500 mM glucose, 10 mM MgCl₂, suspended in 50 mM KPi buffer(pH 7). Using manual pH adjustment, the pH was maintained throughout thebiotransformation at pH 7. The reactions were carried out either withoutcofactor addition or with addition of 0.1 mM NAD. The variation of thebile salt concentrations as a function of time is shown in FIG. 22.After 2 h, with and without addition of NAD, in each case≥98% of product(12-keto-UDCA) was formed.

Using altogether 17.7 g/L BTM biocatalysts, 100 mM DHCA were convertedto an extent of 98% to 12-keto-UDCA.

Reaction Example 13: Two-Step Reduction of Dehydrocholic Acid withParallel Use of Two Different Whole-Cell Biocatalysts

In this example, two whole-cell biocatalysts were used instead of onewhole-cell biocatalyst. For this purpose, an NADP-specific FDH fromMycobacterium vaccae and an NADPH-specific 7β-HSDH from Collinsellaaerofaciens were expressed in one of these biocatalysts, whereas anNAD-dependent FDH from Mycobacterium vaccae and an NADH-dependent3α-HSDH from Comamonas testosteroni were expressed in the otherbiocatalyst.

In the present example, concretely the vector pF(G)r7(A), whichcomprises genes for an NADP-specific FDH from Mycobacterium vaccae andfor an NADPH-specific 7β-HSDH from Collinsella aerofaciens, and thevector pFr3, which comprises genes for an NAD-dependent FDH fromMycobacterium vaccae and for an NADH-dependent 3α-HSDH from Comamonastestosteroni, were used. The vectors pF(G)r7(A) and pFr3 are shown inFIG. 23 a and b. FIG. 24 shows a reaction scheme with possible routesand products. However, the invention is not restricted to these twostated vectors, but can comprise all conceivable vectors that comprisegenes for a 7β-HSDH and a suitable cofactor regeneration enzyme incombination with vectors that comprise genes for a 3α-HSDH and asuitable cofactor regeneration enzyme.

13.1 Whole-Cell Reduction of DHCA Using Two Biocatalysts in DifferentMixture Ratios

The whole-cell reduction of DHCA to 12-keto-UDCA with different mixtureratios of the biocatalysts E. coli BL49 pF(G)r7(A) and E. coli BL49 pFr3is shown in the following example. In this case, a total amount ofbiocatalyst of 17.7 g L⁻¹ _(BTM) was used in each case for threebatches. However, different proportions of the two biocatalysts wereused in the batches. These are

-   -   8.85 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 8.85 g L⁻¹        _(BTM) E. coli BL49 pFr3    -   10.33 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 7.38 g L⁻¹        _(BTM) E. coli BL49 pFr3    -   11.80 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 5.90 g L⁻¹        _(BTM) E. coli BL49 pFr3        These biocatalysis reactions were carried out at a working        volume of 20 mL. Further constituents of the batches were: 70 mM        DHCA, 500 mL sodium formate, 26% glycerol, 50 mM MgCl₂,        suspended in 50 mL potassium phosphate buffer (pH 6.5). The        reactions were carried out for 24 h. The proportions of bile        salts after 24 h are shown in FIG. 25.

In the preparation with 8.85 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and8.85 g L⁻¹ _(BTM) E. coli BL49 pFr3, 79% 12-keto-UDCA, 4%3,12-diketo-UDCA and 17% 7,12-diketo-UDCA are formed, in the preparationwith 10.33 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 7.38 g L⁻¹ _(BTM) E.coli BL49 pFr3, 87% 12-keto-UDCA, 9% 3,12-diketo-UDCA and 4%7,12-diketo-UDCA are formed and in the preparation with 11.80 g L⁻¹_(BTM) E. coli BL49 pF(G)r7(A) and 5.90 g L⁻¹ _(BTM) E. coli BL49 pFr3,71% 12-keto-UDCA and 29% 3,12-diketo-UDCA are formed. It can be seenfrom the proportions of the intermediates formed that in the case of thepreparation with 8.85 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 8.85 gL⁻¹ _(BTM) E. coli BL49 pFr3 the reaction of 3α-HSDH takes place at anincreased rate, as in this case a high proportion of the intermediate7,12-diketo-UDCA is formed, whereas in the case of the preparation of11.80 g L⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 5.90 g L⁻¹ _(BTM) E. coliBL49 pFr3 the reaction of 7β-HSDH takes place at an increased rate andaccordingly a high proportion of the intermediate 3,12-diketo-UDCA isformed. In the preparation with 10.33 g L⁻¹ _(BTM) E. coli BL49pF(G)r7(A) and 7.38 g L⁻¹ _(BTM) E. coli BL49 pFr3, the reactions of7β-HSDH and of 3α-HSDH are adjusted so that both occur at a roughlyequal rate, as can be seen from the almost uniform formations ofintermediates. As a result, the highest proportion of the product12-keto-UDCA (87%) can be formed with this preparation.

This example shows that the rate of the individual reaction steps can beinfluenced by adjusting the proportions of the biocatalysts used.

13.1 Whole-Cell Reduction of DHCA Using Two Biocatalysts at the 1 LScale

Through process control, using the biocatalysts E. coli BL49 pF(G)r7(A)and E. coli BL49 pFr3, product formation could be increased relative tothe milliliter scale. The biotransformation was set up in a 1 Lbioreactor in the following reaction conditions: 90 mM DHCA, with 8.85 gL⁻¹ _(BTM) E. coli BL49 pF(G)r7(A) and 8.85 g L⁻¹ _(BTM) E. coli BL49pFr3, 500 mM ammonium formate, 26% (v/v) glycerol, 50 mM MgCl₂,suspended in 50 mM KPi buffer (pH 6.5). Using pH adjustment with formicacid, the pH was maintained at pH 6.5 throughout the biotransformation.The variation of the bile salt concentrations as a function of time isshown in FIG. 26. After 20 h, 99.4% product (12-keto-UDCA) had formed,and only a total of 0.6% of the intermediates 3,12-diketo-UDCA and7,12-diketo-UDCA was detectable.

Using the biocatalysts according to the invention, with the use of atotal of 17.7 g L⁻¹ _(BTM) biocatalysts, 90 mM DHCA could be convertedto an extent of 99.5% to 12-keto-UDCA.

Assignment of SEQ ID NOs:

SEQ ID NO: Description Type 1 7β-HSDH NS 2 7β-HSDH AS 3 Primer S7beta_rev_HindIII NS 4 Primer NS 5 3α-HSDH (C. testosteroni) NS 63α-HSDH (C. testosteroni) AS 7 3α-HSDH (R. norvegicus) NS 8 3α-HSDH (R.norvegicus) AS 9 Primer 7beta_mut_G39A_fwd NS 10 Primer7beta_mut_G39A_rev NS 11 Primer 7beta_mut_G39S_fwd NS 12 Primer7beta_mut_G39S_rev NS 13 Primer 7beta_fwd_EcoRI NS 14 FDH D221G NS 15FDH D221G AS 16 Primer 3alpha_fwd_HindIII NS 17 Primer 3alpha_rev_NotINS 18 pET22a FDH D221G 7β-HSDH NS 19 FDH D221G AS 20 7beta-HSDH AS 21pCOLA(mod) 3α-HSDH NS 22 3α-HSDH AS 23 Primer fdh_for NS 24 Primerfdh_rev NS 25 7α-HSDH NS 26 7α-HSDH AS 27 Primer 467|468a-IBS NS 28Primer 467|468a-EBS1d NS 29 Primer 467|468a-EBS2 NS 30 Primer mt1 NS 31Primer NI_fdh_R NS 32 Primer 7alpha-ko-check_fwd NS 33 Primer7alpha-ko-check_rev NS 34 FDH D221G with deletion and His-Tag NS 35 FDHD221G with deletion and His-Tag AS 36 FDH wild type, M. vaccae AS 377β-HSDH G39D AS 38 7β-HSDH G39D/R40L AS 39 7β-HSDH G39D/R40I AS 407β-HSDH G39D/R40V AS 41 Primer for G39D (7beta mut G39D fwd) NS 42Primer for G39D (7beta mut G39D rev) NS 43 Primer for G39D R40L (7betamut G39D NS R40L fwd) 44 Primer 3alphamut_AntiMid_rev NS 45 Primer forG39D R40I (7beta mut G39D NS R40I fwd) 46 Primer 7beta mut G39D R40V fwdNS 47 GDH, B. subtilis NS 48 GDH B. subtilis AS 49 Vector pFr7(D) NS 50Vector pFr7(DL) NS 51 Vector pFr7(DI) NS 52 Vector pFr7(DV) NS 53 PCRPrimer “G39D for” NS 54 PCR Primer “G39D_rev” NS 55 PCR Primer“G39D/R40I_for” NS 56 PCR Primer “G39D/R40I_rev” NS 57 PCR Primer“R40D_for” NS 58 PCR Primer “R40D_rev” NS 59 PCR Primer “R40D/R41I_for”NS 60 PCR Primer “R40D/R41I_rev” NS 61 PCR Primer “DIN_for” NS 62 PCRPrimer “DIN_rev” NS 63 Plasmid pFr3T7(D), FIG. 15 NS 64 PlasmidpF(G)r7(A)r3, FIG. 17a NS 65 Plasmid pF(G)r7(S)r3, FIG. 17b NS 66Plasmid p3T7(A)rG, FIG. 20 NS 67 Plasmid p7(A)T3rG, FIG. 21 NS 68Plasmid pF(G)r7(A), FIG. 23a NS 69 Plasmid pFr3, FIG. 23b NS AS = aminoacid sequence NS = nucleic acid sequenceReference is made expressly to the disclosure of the documents mentionedherein.

The invention claimed is:
 1. A 7β-HSDH mutant, which catalyzes at leastthe stereospecific enzymatic reduction of a 7-ketosteroid to thecorresponding 7-hydroxysteroid, wherein the mutant has at least 90%sequence identity to SEQ ID NO:2 and at least one mutation in thesequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ IDNO:2, wherein the mutation is selected from the group consisting of: a)the single mutation G39X₁; b) the single mutation R40X₂; c) the doublemutation G39X₁ R40X₂ d) the double mutation R40X₂ R41X₃; e) the doublemutation G39X₁ R41X₃; and f) the triple mutation G39X₁ R40X₂ R41X₃;wherein each of X₁, X₂ and X₃ can be the same or different and standsfor mutated amino acid residues.
 2. The 7β-HSDH mutant of claim 1,wherein the mutant has decreased substrate inhibition for a7-ketosteroid substrate.